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Marco BORSARI

Professore Associato
Dipartimento di Scienze Chimiche e Geologiche - Sede Dipartimento di Scienze Chimiche e Geologiche

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Pubblicazioni

2024 - EFFICIENT ELECTROCATALYTIC H2 PRODUCTION BY IMMOBILIZED Co(III)-MYOGLOBIN [Articolo su rivista]
Meglioli, Mirco; DI ROCCO, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Sola, Marco; Battistuzzi, Gianantonio; Borsari, Marco
abstract

The thermodynamics and kinetics of heterogeneous electron transfer (ET) for Co-substituted horse myoglobin (Co-Mb) and its derivatives with ammonia and imidazole as heme axial ligands were studied with cyclic voltammetry on a pyrolytic graphite electrode along with their ability to mediate the electrocatalytic production of H2 . All the proteins experience a non-diffusive electrochemical regime as electrode-bound species. The adsorbed Co-Mb construct was found to carry out the electrocatalytic reduction of water protons to H2 with a good efficiency under anaerobic conditions thus yielding a simple and tunable system for H2 production. Replacement of H2O as Co axial ligand by ammonia and imidazole significantly lowers the catalytic currents for H3O+/H2O reduction to H2. The E°’ values of the Co(III)/Co(II) redox couple for all species are mainly determined by the enthalpic contribution. Differences were found in the kinetics of ET for the different protein adducts due to changes in the activation enthalpies. However, all species share the same distance of about 14 Å from the electrode surface to the Co(III)/Co(II) center determined using the Marcus model, consistent with a non-denaturing adsorption of the protein.

2024 - Quantum Spin Coherence and Electron Spin Distribution Channels in Vanadyl-containing Lantern Complexes [Articolo su rivista]
Imperato, Manuel; Nicolini, Alessio; Borsari, Marco; Briganti, Matteo; Chiesa, Mario; Liao, Yu-Kai; Ranieri, Antonio; Raza, Arsen; Salvadori, Enrico; Sorace, Lorenzo; Cornia, Andrea
abstract

We herein investigate the heterobimetallic lantern complexes [PtVO(SOCR)4] as charge neutral electronic qubits based on vanadyl complexes (S = 1/2) with nuclear spin-free donor atoms. The derivatives with R = Me (1) and Ph (2) give highly resolved X-band EPR spectra in frozen CH2Cl2/toluene solution, which evidence the usual hyperfine coupling to the 51V nucleus (I = 7/2) and an additional superhyperfine interaction with the I = 1/2 nucleus of the 195Pt isotope (natural abundance ca. 34%). DFT calculations ascribe the spin-density delocalization on the Pt2+ ion to a combination of π and δ pathways, with the former representing the predominant channel. Spin relaxation measurements in frozen CD2Cl2/toluene-d8 solution between 90 and 10 K yield Tm values (1-6 μs in 1 and 2-11 μs in 2) which match or even outperform those of known vanadyl-based qubits in similar matrices. Coherent spin manipulations indeed prove possible at 70 K, as shown by the observation of Rabi oscillations in nutation experiments. The results indicate that the heavy Group 10 metal is not detrimental to the coherence properties of the vanadyl moiety and that Pt-VO lanterns can be used as robust spin-coherent building blocks in materials science and quantum technologies.

2023 - A remarkably unsymmetric hexairon core embraced by two high-symmetry tripodal oligo-α-pyridylamido ligands [Articolo su rivista]
Nicolini, Alessio; Pankratz, Trey C.; Borsari, Marco; Clérac, Rodolphe; Ranieri, Antonio; Rouzières, Mathieu; Berry, John F.; Cornia, Andrea
abstract

Oligo-α-pyridylamides offer an appealing route to polyiron complexes with short Fe-Fe separations and large room temperature magnetic moments. A derivative of tris(2‐aminoethyl)amine (H6tren) containing three oligo-α-pyridylamine branches and thirteen nitrogen donors (H6L) reacts with [Fe2(Mes)4] to yield an organic nanocage built up by two tripodal ligands with interdigitated branches (HMes = mesitylene). The nanocage has crystallographic D3 symmetry but hosts a remarkably unsymmetric hexairon-oxo core, with a central Fe5(μ5-O) square pyramid, two oxygen donors bridging basal sites, and an additional Fe center residing in one of the two tren-like pockets. Bond Valence Sum (BVS) analysis, Density Functional Theory (DFT) calculations, and electrochemical data were then used to establish the protonation state of oxygen atoms and the formal oxidation states of the metals. To this purpose, a specialized set of BVS parameters was devised for Fe2+-N3- bonds with nitrogen donors of oligo-α-pyridylamides. This allowed us to formulate the compound as [Fe6O2(OH)(H3L)L], with nominally four FeII and two FeIII ions. Mössbauer spectra indicate that the compound contains two unique FeII sites, identified as a pair of closely spaced hydroxo-bridged metal ions in the central Fe5(μ5-O) pyramid, and a substantially valence-delocalized FeII2FeIII2 unit. Broken-symmetry DFT calculations predict strong ferromagnetic coupling between the two iron(II) ions, leading to a local S = 4 state that persists to room temperature and explaining the large magnetic moment measured at 300 K. The compound behaves as a single-molecule magnet, with magnetization dynamics detectable in zero static field and dominated by an Orbach-like mechanism with Ueff/kB = 49(2) K and τ0 = 4(2)·10−10 s.

2023 - Effects of removal of the axial methionine heme ligand on the binding of S. cerevisiae iso-1 cytochrome c to cardiolipin. [Articolo su rivista]
Paradisi, Alessandro; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Battistuzzi, Gianantonio
abstract

The cleavage of the axial S(Met)-Fe bond in cytochrome c (cytc) upon binding to cardiolipin (CL), a glycerophospholipid of the inner mitochondrial membrane, is one of the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its pro-apoptotic function. In this work, UV-VIS, CD, MCD and fluorescence spectroscopies were used to address the role of the Fe−M80 bond in controlling the cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S. cerevisiae iso-1 cytc (ycc) to CL liposomes in comparison with the wt protein [Paradisi et al. J. Biol. Inorg. Chem. 25 (2020) 467–487]. The results show that the integrity of the six-coordinate heme center along with the distal heme site containing the Met80 ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe-S(Met80) bond has a little impact on the mechanism of ycc-CL interaction, although it results in an increased heme accessibility to solvent and a reduced structural stability of the protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios compared to wt ycc, possibly due to the lift of some constraints to the insertion of the CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the dependence on the CL-to-cytc mixing scheme displayed by the wt species

2023 - Hydrogen Peroxide Induces Heme Degradation and Protein Aggregation in Human Neuroglobin: Roles of the Disulfide Bridge and the H-bonding in the Distal Heme Cavity [Articolo su rivista]
Di Rocco, G.; Bernini, F.; Battistuzzi, G.; Ranieri, A.; Bortolotti, C. A.; Borsari, M.; Sola, M.
abstract

In this study, human neuroglobin (hNgb) was found to undergo H2O2-induced breakdown of the heme center at a much slower rate than other globins, namely in the timescale of hours against minutes. We studied how the rate of the process is affected by the Cys46/Cys55 disulfide bond and the network of noncovalent interactions in the distal heme side involving Tyr44, Lys67, the His64 heme iron axial ligand and the heme propionate-7. The rate is increased by the Tyr44 to Ala and Phe mutations, however the rate is lowered by Lys67 to Ala swapping. The absence of the disulfide bridge slows down the reaction further. Therefore, the disulfide bond-controlled accessibility of the heme site and the residues at position 44 and 67 affect the activation barrier of the reaction. Wild-type and mutated species form -amyloid aggregates in the presence of H2O2 producing globular structures. Furthermore, the C46A/C55A, Y44A, Y44F and Y44F/C46A/C55A variants yield potentially harmful fibrils. Finally, the nucleation and growth kinetics for the aggregation of the amyloid structures can be successfully described by the Finke-Watzky model.

2023 - Near Infrared and UV-Visible Spectroscopy Coupled with Chemometrics for the Characterization of Flours from Different Starch Origins [Articolo su rivista]
Pellacani, Samuele; Borsari, Marco; Cocchi, Marina; D'Alessandro, Alessandro; Durante, Caterina; Farioli, Giulia; Strani, Lorenzo
abstract

2022 - Assessing the functional and structural stability of the Met80Ala mutant of cytochrome c in dimethylsulfoxide [Articolo su rivista]
DI ROCCO, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio
abstract

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and bi-osensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cyto-chrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscop-ically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the mod-ification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physi-ological milieu of cytochrome c.

2022 - Detection of Neurofilament Light Chain with Label-Free Electrolyte-Gated Organic Field-Effect Transistors [Articolo su rivista]
Solodka, K.; Berto, M.; Ferraro, D.; Menozzi, C.; Borsari, M.; Bortolotti, C. A.; Biscarini, F.; Pinti, M.
abstract

Neurofilaments are structural scaffolding proteins of the neuronal cytoskeleton. Upon axonal injury, the neurofilament light chain (NF-L) is released into the interstitial fluid and eventually reaches the cerebrospinal fluid and blood. Therefore, NF-L is emerging as a biomarker of neurological disorders, including neurodegenerative dementia, Parkinson's disease, and multiple sclerosis. It is challenging to quantify NF-L in bodily fluids due to its low levels. This work reports the detection of NF-L in aqueous solutions with an organic electronic device. The biosensor is based on the electrolyte-gated organic field-effect transistor (EGOFET) architecture and can quantify NF-L down to sub-pM levels; thanks to modification of the device gate with anti-NF-L antibodies imparted with potentially controlled orientation. The response is fitted to the Guggenheim–Anderson–De Boer adsorption model to describe NF-L adsorption at the gate/electrolyte interface, to consider the formation of a strongly adsorbed protein layer bound to the antibody and the formation of weakly bound NF-L multilayers, an interpretation which is also backed up by morphological characterization via atomic force microscopy. The label-free, selective, and rapid response makes this EGOFET biosensor a promising tool for the diagnosis and monitoring of neuronal damages through the detection of NF-L in physio-pathological ranges.

2022 - Thermodynamics and Kinetics of Electron Transfer of 2 Electrode-Immobilized Small Laccase from Streptomyces coelicolor [Articolo su rivista]
DI ROCCO, Giulia; Battistuzzi, Gianantonio; Ranieri, Antonio; Bortolotti, Carlo Augusto; Borsari, Marco; Sola, Marco
abstract

The thermodynamic and kinetic properties for the heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccin-imide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°’ values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can respectively be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°’ values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pKa values of pKa1ox =5.1, pKa1red=7.5, pKa2ox=8.4, pKa2red=10.9, pKa2ox=8.9, pKa2red=11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°’ value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and me-30 dium conditions.

2021 - Chemical and biochemical thermodynamics reunification (IUPAC Technical Report) [Articolo su rivista]
Iotti, S.; Sabatini, A.; Borsari, M.; Moss, G. P.
abstract

According to the 1994 IUBMB-IUPAC Joint Commission on Biochemical Nomenclature (JCBN) on chemical and biochemical reactions, two categories of thermodynamics, based on different concepts and different formalisms, are established: (i) chemical thermodynamics, which employ conventional thermodynamic potentials to deal with chemical reactions [1-3]; and (ii) biochemical thermodynamics, which employ transformed thermodynamic quantities to deal with biochemical reactions based on the formalism proposed by Alberty [4-7]. We showed that the two worlds of chemical and biochemical thermodynamics, which so far have been treated separately, can be reunified within the same thermodynamic framework. The thermodynamics of chemical reactions, in which all species are explicitly considered with their atoms and charge balanced, are compared with the transformed thermodynamics generally used to treat biochemical reactions where atoms and charges are not balanced. The transformed thermodynamic quantities suggested by Alberty are obtained by a mathematical transformation of the usual thermodynamic quantities. The present analysis demonstrates that the transformed values for ΔrG′0 and ΔrH′0 can be obtained directly, without performing any transformation, by simply writing the chemical reactions with all the pseudoisomers explicitly included and the elements and charges balanced. The appropriate procedures for computing the stoichiometric coefficients for the pseudoisomers are fully explained by means of an example calculation for the biochemical ATP hydrolysis reaction. It is concluded that the analysis reunifies the “two separate worlds” of conventional thermodynamics and transformed thermodynamics.

2021 - Copper-catalyzed ARGET ATRP of styrene from ethyl α-haloisobutyrate in EtOAc/EtOH, using ascorbic acid/Na2CO3 as reducing system [Articolo su rivista]
Borsari, M.; Braidi, N.; Buffagni, M.; Ghelfi, F.; Parenti, F.; Porcelli, N.; Serafini, G.; Isse, A. A.; Bonifaci, L.; Cavalca, G.; Longo, A.; Morandini, I.; Pettenuzzo, N.
abstract

Atom transfer radical polymerization (ATRP) is one of the most powerful techniques to synthesize precisely tailored polymers and macrostructures. Activators regenerated by electron transfer (ARGET) ATRP was developed as a “green” strategy to decrease the load of the metal catalyst. However, ARGET ATRP usually uses not-so-green reducing agents (e.g. Sn2+) or expensive and industrially impractical procedures. For these reasons, we report an ARGET ATRP of styrene with various carbonates as reducing agents, both alone and paired with ascorbic acid (AA), using monofunctional initiators and Cl or Br as exchanging atoms. The solvent mixture is composed of the green combination of EtOAc and EtOH, even in the presence of modest amounts of H2O. Cyclic voltammetry was used to evaluate the effect of H2O on the catalyst, as well as the absence of AA. The system returned high yields and low dispersities with low amounts of catalyst (~60 ppm) and moderate reaction times. Excellent results were obtained only with the Cl-ATRP system. NMR spectroscopy and kinetic analyses proved the livingness of the final polymer chains.

2021 - Electron Transfer and Electrocatalytic Properties of the Immobilized Met80Ala Cytochrome c Variant in DMSO [Articolo su rivista]
DI ROCCO, Giulia; Bighi, Beatrice; Borsari, Marco; Bortolotti, Carlo Augusto; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract

The electrode-immobilized Met80Ala variant of yeast iso-1 cytochrome c in mixed water/dimethylsulfoxide (DMSO) solutions up to 60 % v/v DMSO shows thermodynamic and kinetic parameters of electron exchange and electrocatalytic properties towards O2 reduction fully comparable to those in water. This is the result of moderate protein conformational changes thanks to immobilization that, to a certain extent, preserves protein structure, possibly due to the constraints on protein mobility/flexibility induced by the electrostatic interactions with the electrode-coating SAM. Upon increasing the DMSO content of the mixed solution beyond 60 %, a much larger perturbation occurs that leads to the progressive loss of the electrocatalytic ability. Therefore, under these conditions, the organic solvent remarkably affects the structure and properties of the protein probably involving major conformational changes or even the replacement of the 6th axial hydroxide ligand of the heme iron with a strong protein ligand, possibly a lysine residue.

2021 - Gaseous heptanethiol removal by a fe3+-phenanthroline-kaolinite hybrid material [Articolo su rivista]
Bernini, F.; Castellini, E.; Bighi, B.; Borsari, M.; Malferrari, D.; Brigatti, M. F.
abstract

Kaolinite functionalized by the μ-oxo Fe3+-phenanthroline complex (Fe+3Phen) was selected to test its ability to efficiently remove and store gaseous heptanethiol (HPT). Spectroscopic techniques, elemental analysis, and thermal analysis coupled with evolved gas mass spectrometry were employed to characterize the material before and after the exposure to the gas and to define the adsorption process. The amount of HPT trapped by the functionalized kaolinite after 60 days is 0.10940 moles per 100 g of kaolinite which, considering the amount of adsorbed Fe+3Phen (0.00114 moles per 100 g of kaolinite), means a thiol/Fe3+Phen molar ratio of about 100:1, a value much higher than those found in the past for Fe+3Phen functionalized montmorillonite and sepiolite. In addition, the process was found to be efficient also beyond 60 days. This significant removal of the smelly gas was explained by considering a continuous catalytic activity of Fe3+ toward the oxidation of thiol to disulfide.

2021 - How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing [Articolo su rivista]
Ranieri, Antonio; Borsari, Marco; Casalini, Stefano; Di Rocco, Giulia; Sola, Marco; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio
abstract

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.

2021 - Molecular structure and ammonia gas adsorption capacity of a Cu(II)-1,10-phenanthroline complex intercalated in montmorillonite by DFT simulations [Articolo su rivista]
Sainz-Diaz, C. I.; Castellini, E.; Escamilla-Roa, E.; Bernini, F.; Malferrari, D.; Brigatti, M. F.; Borsari, M.
abstract

A hydrated complex of 1,10-phenanthroline with Cu2+ cation was intercalated in the interlayer space of montmorillonite. This intercalation occurs initially by through a cation exchange mechanism in which the charge of the complex cation compensates the excess of the negative charge of the interlayer, then, once the cation exchange capacity (CEC) value has been reached, by direct adsorption of the sulfate salt of this complex (i.e. the cation together with its sulfate counterion). This material has showed interesting entrapping properties of gaseous phases and a peculiar chemical reactivity. However, the complete characterization and explanation of the formation of these materials is difficult with only experimental techniques. Hence, we used computational methods at atomic level to know how are the molecular structure of these complexes and their adsorption capacity of ammonia inside the interlayer confined space of montmorillonite for a better understanding of the experimental behaviour. First Principles calculations were performed based on Density Functional Theory (DFT). The intercalation of the phenanthroline-Cu(II) complex inside the nanoconfined interlayer of montmorillonite is energetically favourable in the relative proportion observed experimentally, being a cation exchange process. The further adsorption of the sulfate salt of the phenanthroline-Cu complex is also energetically possible. The adsorption of ammonia molecules in these montmorillonite-phenanthroline-Cu complexes was also favourable according with experimental behaviour.

2021 - Physical insights from the Frumkin isotherm applied to electrolyte gated organic transistors as protein biosensors [Articolo su rivista]
Manco Urbina, Pamela Allison; Berto, Marcello; Greco, Pierpaolo; Sensi, Matteo; Borghi, Simone; Borsari, Marco; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract

2021 - Pseudoperoxidase activity, conformational stability and aggregation propensity of the His98Tyr myoglobin variant. Implications for the onset of myoglobinopathy. [Articolo su rivista]
Hofbauer, Stefan; Pignataro, Marcello; Borsari, Marco; Bortolotti, Carlo; Di Rocco, Giulia; Ravenscroft, Gianina; Furtmüller, Paul; Obinger, Christian; Sola, Marco; Battistuzzi, Gianantonio
abstract

The autosomal dominant striated muscle disease myoglobinopathy is due to the single point mutation His98Tyr in human myoglobin (MB) [Olivè et al. Nat. Comm, 2019, 10, 1396], the heme-protein responsible for binding, storage and controlled release of O2 in striated muscle. In order to understand the molecular bases of this disease, a comprehensive biochemical and biophysical study on wt MB and the variant H98Y has been performed. Although only small differences exist between the active site architectures of the two proteins, the mutant exhibits an (i) increased reactivity towards hydrogen peroxide, (ii) a higher tendency to form high-molecular weight aggregates and (iii) is more prone to heme bleaching, possibly as a consequence of the observed H2O2-induced formation of the Tyr98 radical close to the metal center. These effects add to the impaired oxygen binding capacity and faster heme dissociation of the H98Y variant compared to wt MB. As the above effects result from bond formation/cleavage events occurring at the distal and proximal heme sites, it appears that the molecular determinants of the disease are localized there. These findings set the bases for clarifying the onset of the cascade of chemical events that are responsible for the pathological symptoms of myoglobinopathy.

2021 - Self-Assembled Structures from Solid Cd(II) Acetate in Thiol/Ethanol Solutions: a Novel Type of Organic Chemical Garden [Articolo su rivista]
Bernini, Fabrizio; Castellini, Elena; Sebastianelli, Lorenzo; Bighi, Beatrice; Ignacio Sainz-Díaz, Claro; Mucci, Adele; Malferrari, Daniele; Ranieri, Antonio; Franca Brigatti, Maria; Borsari, Marco
abstract

Cd(II) acetate in tablet form, immersed in ethanol solutions with a high concentration of heptanethiol (30-80 mM) was able to develop self-assembled sail-shaped structures. The solution in which the self-assembled structure is formed is totally organic (both solute and solvent), thus representing a unique case among organic Chemical Gardens. The constituting material for this new Chemical Garden (CG) is made of a single phase, namely a cadmium heptanethiolate with a microcrystalline structure consisting of a central cluster [CdnSn]. The morphological and structural features were studied using different techniques (SEM-EDS, elemental analysis, FT-IR, 113Cd MAS NMR, XRPD). A crystalline cell for the Cd(II) heptanethiolate cluster was obtained that completely differs from that of the solid phase precipitated by mixing an ethanol solution of Cd(II) acetate with heptanethiol. It follows that the conditions under which the formation of Cd(II) thiolate occurs (slowly from solid, quickly from solution) play a critical role in determining the nature and structure of the precipitating phase.

2021 - Tetrairon(II) extended metal atom chains as single-molecule magnets [Articolo su rivista]
Nicolini, A; Affronte, M; SantaLucia, Dj; Borsari, M; Cahier, B; Caleffi, M; Ranieri, A; Berry, Jf; Cornia, A
abstract

Iron-based extended metal atom chains (EMACs) are potentially high-spin molecules with axial magnetic anisotropy and thus candidate single-molecule magnets (SMMs). We herein compare the tetrairon(II), halide-capped complexes [Fe4(tpda)3Cl2] (1Cl) and [Fe4(tpda)3Br2] (1Br), obtained by reacting iron(II) dihalides with [Fe2(Mes)4] and N2,N6-di(pyridin-2-yl)pyridine-2,6-diamine (H2tpda) in toluene, under strictly anhydrous and anaerobic conditions (HMes = mesitylene). Detailed structural, electrochemical and Mossbauer data are presented along with direct-current (DC) and alternating-current (AC) magnetic characterizations. DC measurements revealed similar static magnetic properties for the two derivatives, with chiMT at room temperature above that for independent spin carriers, but much lower at low temperature. The electronic structure of the iron(II) ions in each derivative was explored by ab initio (CASSCF-NEVPT2-SO) calculations, which showed that the main magnetic axis of all metals is directed close to the axis of the chain. The outer metals, Fe1 and Fe4, have an easy-axis magnetic anisotropy (D = -11 to -19 cm-1, |E/D| = 0.05-0.18), while the internal metals, Fe2 and Fe3, possess weaker hard-axis anisotropy (D = 8-10 cm-1, |E/D| = 0.06-0.21). These single-ion parameters were held constant in the fitting of DC magnetic data, which revealed ferromagnetic Fe1-Fe2 and Fe3-Fe4 interactions and antiferromagnetic Fe2-Fe3 coupling. The competition between super-exchange interactions and the large, noncollinear anisotropies at metal sites results in a weakly magnetic non-Kramers doublet ground state. This explains the SMM behavior displayed by both derivatives in the AC susceptibility data, with slow magnetic relaxation in 1Br being observable even in zero static field.

2021 - The Copper Chemical Garden as a Low Cost and Efficient Material for Breaking Down Air Pollution by Gaseous Ammonia [Articolo su rivista]
Castellini, Elena; Bernini, Fabrizio; Sebastianelli, Lorenzo; Bighi, Beatrice; Ignacio Sainz-Diaz, Claro; Mucci, Adele; Malferrari, Daniele; Ranieri, Antonio; Gorni, Giulio; Marini, Carlo; Brigatti, Maria Franca; Borsari, Marco
abstract

Chemical garden (CG) from copper(II) sulfate, nitrate and chloride (CG CuSO4, CG Cu(NO3)2, CG CuCl2) were grown and characterized from the structural and compositional point of view using scanning electron microscopy, X-ray powder diffraction, elemental analysis, thermogravimetric analyses coupled with mass spectrometry, and DR (Diffuse Reflectance) UV-Vis-Nir spectroscopy. The main crystalline phases, controlled by the anion of the starting salt, were brochantite and kobyashevite for CG CuSO4, gerhardtite, rouaite and anthonyite for CG Cu(NO3)2, and atacamite for CG CuCl2. The materials were then exposed to ammonia vapors to test the effectiveness of their entrapping property. All materials proved to be very efficient and rapid in the uptake of ammonia which invariably results in the formation of a Cu(II)/NH3 complex. However, after a few tens of minutes, CG Cu(NO3)2 and CG CuCl2 release water and get wet, thereby resulting unsuitable for applications. Only CG CuSO4 keeps dry for at least 25 hours. This makes it a valid candidate for building devices for trapping ammonia and possibly other gases capable of interacting with Cu (II). The entrapment of ammonia by this material was also characterized by 1H and 29Si MAS-NMR XAS spectroscopies.

2021 - The Enthalpic and Entropic Terms of the Reduction Potential of Metalloproteins: Determinants and Interplay [Articolo su rivista]
Di Rocco, G.; Battistuzzi, G.; Borsari, M.; Bortolotti, C. A.; Ranieri, A.; Sola, M.
abstract

Splitting the reduction potential of electron transport (ET) proteins and redox metalloenzymes into the enthalpic and entropic contributions is an insightful practice to gaining insight into the molecular determinants of the thermodynamic propensity of the metal center to accept or release electrons. The strict control of such propensity is essential for the functioning of the electron transport chains in bioenergetics and the organized network of the countless reactions of the redox metabolism in all organisms. Here, the first comprehensive overview is offered on the thermodynamic data obtained in the last three decades for the main classes of ET species, namely c-type cytochromes and proteins containing T1 copper and iron-sulfur centers, along with some heme metalloenzymes. These families show many common features in the balance of the enthalpic and entropic terms, which will be brought to light. The enthalpic terms related to ligation features in the first coordination sphere of the metal and weak binding and electrostatics in the surrounding matrix do count a lot in this balance. Reduction entropy is much less important that it would appear from the raw thermodynamic data, particularly for electron transport (ET) metalloproteins. This is due to reduction-induced solvent-related molecular events which dominate the measured entropy changes but affect much less the reduction free energy due to the compensatory effects of the associated enthalpic terms (a phenomenology known as enthalpy-entropy compensation, EEC). Thus the entropy changes seldom exert a real influence on the E°’ of metalloredox proteins; this is restricted to metal sites subjected to reduction-induced protein-based changes in the accessible configurational microstates. It follows that in most cases, especially for ET species, the E° changes due to point mutations, ligand binding and charge changes have and ultimate enthalpic origin. Hence, they should be accounted for with coordination chemistry and electrostatics notions. Only if they don’t, protein-based entropic effects could play a role. In this review, we go through the data gathered for the main classes of ET species and heme enzymes that brought us to this conclusion.

2020 - A new material based on montmorillonite and Cu(II)-phenanthroline complex for effective capture of ammonia from gas phase [Articolo su rivista]
Castellini, E.; Malferrari, D.; Bernini, F.; Bighi, B.; Mucci, A.; Sainz-Diaz, I. C.; Serrano, A.; Castro, G. R.; Brigatti, M. F.; Borsari, M.
abstract

The intercalation of [Cu(Phen)(H2O)2]2+ (CuPhen) in montmorillonite (Mt) produces a stable hybrid material that is very efficient in removing NH3 from gas phase even at extremely low pressures. The process was studied by elemental analysis, X-ray powder diffraction, thermal analysis coupled with evolved gas mass spectrometry and DR UV–Vis, NMR and X-ray absorption spectroscopy. The adsorption of CuPhen on Mt consists of two consecutive steps. During the first one, CuPhen intercalates alone into Mt through a cation exchange process, afterwards CuPhen and SO42− ions entry jointly into the mineral interlayer. The two-steps adsorption process is described by a VI-type isotherm, successfully fitted by two independent Frumkin isotherms. NH3 trapping is long-lasting, easy, fast even at extremely low gas pressure and reversible under mild conditions. Mt containing CuPhen always results well performant in removing ammonia from gas phase, but an appreciably higher adsorption capacity of NH3 is obtained when SO42− ion is absent from the interlayer. This hybrid montmorillonite is thus a promising material to be used in industrial or environmental contexts, as an efficient air-cleaner.

2020 - Adsorbing surface strongly influences the pseudoperoxidase and nitrite reductase activity of electrode-bound yeast cytochrome c. The effect of hydrophobic immobilization. [Articolo su rivista]
Lancellotti, Lidia; Borsari, Marco; Bonifacio, Alois; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Casalini, Stefano; Ranieri, Antonio; Battistuzzi, Gianantonio; Sola, Marco
abstract

The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) and their adducts with cardiolipin immobilized onto a gold electrode coated with a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol were studied through cyclic voltammetry and surface-enhanced resonance Raman spectroscopy (SERRS). The electroactive species - containing a six-coordinate His/His axially ligated heme and a five-coordinate His/- heme stable in the oxidized and reduced state, respectively - and the pseudoperoxidase activity match those found previously for the wt species and are only slightly affected by CL binding. Most importantly, the reduced His/- ligated form of these variants is able to catalytically reduce the nitrite ion, while electrode-immobilized wt ycc and other His/Met heme ligated variants under a variety of conditions are not. Besides the pseudoperoxidase and nitrite reductase functions, which are the most physiologically relevant abilities of these constructs, also axial heme ligation and the equilibria between conformers are strongly affected by the nature - hydrophobic vs. electrostatic - of the non-covalent interactions determining protein immobilization. Also affected are the catalytic activity changes induced by a given mutation as well as those due to partial unfolding due to CL binding. It follows that under the same solution conditions the structural and functional properties of immobilized ycc are surface-specific and therefore cannot be transferred from an immobilized system to another involving different interfacial protein-SAM interactions.

2020 - Binding of S. cerevisiae iso‑1 cytochrome c and its surface lysine‑to‑alanine variants to cardiolipin: charge effects and the role of the lipid to protein ratio [Articolo su rivista]
Paradisi, Alessandro; Bellei, Marzia; Paltrinieri, Licia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Ranieri, Antonio; Borsari, Marco; Sola, Marco; Battistuzzi, Gianantonio
abstract

The interaction of cytochrome c with cardiolipin (CL) is a critical step in the initial stages of apoptosis and is mediated by a positively charged region on the protein surface comprising several lysine residues (site A). Here, the interaction of wt S. cerevisiae cytochrome c (ycc) and its K72A/K73A, K72A/K79A, K73A/K79A and K72A/K73A/K79A variants with CL was studied through UV–Vis and MCD spectroscopies at pH 7 and molecular dynamics (MD) simulations, to clarify the role of the mutated lysines. Moreover, the influence of the lipid to protein ratio on the interaction mechanism was investigated using low (0.5–10) and high (5–60) CL/ycc molar ratios, obtained with small and gradual or large and abrupt CL additions, respectively. Although all proteins bind to CL, switching from the native low-spin His/Met-ligated form to a low-spin bis-His conformer and to a high-spin species at larger CL concentrations, the two schemes of CL addition show relevant differences in the CL/ycc molar ratios at which the various conformers appear, due to differences in the interaction mechanism. Extended lipid anchorage and peripheral binding appear to prevail at low and high CL/ycc molar ratios, respectively. Simultaneous deletion of two or three surface positive charges from Site A does not abolish CL binding, but instead increases protein affinity for CL. MD calculations suggest this unexpected behavior results from the mutation-induced severe weakening of the H-bond connecting the Nε of His26 with the backbone oxygen of Glu44, which lowers the conformational stability compared to the wt species, overcoming the decreased surface electrostatic interaction.

2020 - Electrochemical data on redox properties of human Cofilin-2 and its Mutant S3D [Articolo su rivista]
Pignataro, M.; Di Rocco, G.; Lancellotti, L.; Bernini, F.; Subramanian, K.; Castellini, E.; Bortolotti, C. A.; Malferrari, D.; Moro, D.; Valdrè, G.; Borsari, M.; del Monte, F.
abstract

The reported data are related to a research paper entitled "Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation" [1]. Info about the formation and redox properties of the disulfide bridge of a protein is quite difficult to obtain and only in a few cases was it possible to observe a cyclic voltammetry (CV) signal [2,3]. Human cofilin-2 contains two cysteines (Cys39 and Cys80) which can be oxidized in suitable conditions and form a disulfide bridge [1]. For this purpose, CV measurements were carried out on human cofilin-2 WT and its mutant S3D immobilized on a gold electrode coated by an anionic self-assembled monolayer (SAM), after a pre-oxidation time which was fundamental for observing a CV signal relating to the oxidation/reduction process of the disulfide bridge of the proteins. The data include CV curves obtained with and without electrochemical pre-oxidation and after oxidation with H2O2. In addition, the plot of the cathodic peak current vs. electrochemical pre-oxidation time and the pH dependence of the formal potential (E°’) are reported. The data obtained by CV measurements were used to determine the time required to form the disulfide bridge for the immobilized proteins and, consequently, to observe the CV signal, to calculate the E°’ values and analyse the pH dependence of E°’. The electrochemical data were provided which will be useful for further electrochemical investigations regarding proteins bearing disulfide bridge(s) or cysteines prone to oxidation.

2020 - Flexible Printed Organic Electrochemical Transistors for the Detection of Uric Acid in Artificial Wound Exudate [Articolo su rivista]
Galliani, M.; Diacci, C.; Berto, M.; Sensi, M.; Beni, V.; Berggren, M.; Borsari, M.; Simon, D. T.; Biscarini, F.; Bortolotti, C. A.
abstract

Low-cost, minimally invasive sensors able to provide real-time monitoring of wound infection can enable the optimization of healthcare resources in chronic wounds management. Here, a novel printed organic electrochemical transistors (OECT) biosensor for monitoring uric acid (UA), a bacterial infection biomarker in wounds, is demonstrated in artificial wound exudate. The sensor exploits the enzymatic conversion of UA to 5-hydroxyisourate, catalyzed by Uricase entrapped in a dual-ionic-layer hydrogel membrane casted onto the gate. The sensor response is based on the catalytic oxidation of the hydrogen peroxide, generated as part of the Uricase regeneration process, at the Pt modified gate. The proposed dual membrane avoids the occurrence of nonspecific faradic reactions as, for example, the direct oxidation of UA or other electroactive molecules that would introduce a potentially false negative response. The biosensor is robust and its response is reproducible both in phosphate buffer saline and in complex solutions mimicking the wound exudate. The sensor has a high sensitivity in the range encompassing the pathological levels of UA in wounds (<200 μm) exhibiting a limit of detection of 4.5 μm in artificial wound exudate. All these characteristics make this OECT-based biosensor attractive for wound monitoring interfaced to the patient.

2020 - Interlayer-confined Cu(II) complex as an efficient and long-lasting catalyst for oxidation of H2s on montmorillonite [Articolo su rivista]
Castellini, E.; Bernini, F.; Sebastianelli, L.; Sainz-Diaz, C. I.; Serrano, A.; Castro, G. R.; Malferrari, D.; Brigatti, M. F.; Borsari, M.
abstract

Removal of highly toxic H2S for pollution control and operational safety is a pressing need. For this purpose, a montmorillonite intercalated with Cu(II)-phenanthroline complex [Cu[(Phen)(H2O)2]2+ (Mt-CuPhen) was prepared to capture gaseous H2S under mild conditions. This hybrid material was simple to obtain and demonstrated an outstanding ability to entrap H2S at room temperature, retaining high efficiency for a very long time (up to 36.8 g of S/100 g Mt-CuPhen after 3 months of exposure). Sorbent and H2S uptake were investigated by elemental analysis, X-ray powder diffraction measurements, diffuse reflectance (DR) UV–Vis and infrared spectroscopy, thermal analysis and evolved gas mass spectrometry, scanning electron microscopy equipped with energy-dispersive X-ray spectrometer, and X-ray absorption spectroscopy. The H2S capture was studied over time and a mechanism of action was proposed. The entrapping involves a catalytic mechanism in which [Cu[(Phen)(H2O)2]2+ acts as catalyst for H2S oxidation to S0 by atmospheric oxygen. The low cost and the long-lasting performance for H2S removal render Mt-CuPhen an extremely appealing trap for H2S removal and a promising material for many technological applications.

2020 - Met80 and Tyr67 affect the chemical unfolding of yeast cytochrome c: comparing solution vs. immobilized state [Articolo su rivista]
Paradisi, Alessandro; Lancellotti, Lidia; Borsari, Marco; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract

Urea-induced denaturation of the Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) was studied through variable temperature diffusive cyclic voltammetry and electronic absorption, CD and MCD spectroscopies. The susceptibility to unfolding of both variants - represented by the free energy of unfolding at denaturant infinite dilution, ∆〖G°〗_u^(H_2 O)is greater compared to the species showing an intact Met/His coordination, as observed previously for the same species immobilized onto a functionalized electrode. This is consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Notably, we find that the unfolding propensity and axial heme iron coordination of the present Fe-(S)Met bond-deprived variants is affected by the motional regime of the protein. In particular, electrostatic adsorption onto a negatively charged SAM surface - that would mimic the phospholipidic inner mitochondrial membrane - facilitates unfolding compared to the solution state, especially at room temperature. This finding has a physiological relevance related to the cytochrome c interaction with cardiolipin at the IMM in the early stages of apoptosis. Moreover, while both immobilized variants maintain the His/OH- axial heme iron coordination up to 7 M urea, the same species in solution are subjected to urea-induced replacement of the axial hydroxide ligand by a His ligand. The contribution of the enthalpic and entropic terms to ∆〖G°〗_u^(H_2 O) were found to be opposite (H-S compensation) indicating that unfolding thermodynamics are strongly affected by changes in the hydrogen bonding network in the hydration sphere of the protein.

2020 - Phosphorylated cofilin-2 is more prone to oxidative modifications on Cys39 and favors amyloid fibril formation [Articolo su rivista]
Pignataro, M.; Di Rocco, G.; Lancellotti, L.; Bernini, F.; Subramanian, K.; Castellini, E.; Bortolotti, C. A.; Malferrari, D.; Moro, D.; Valdre, G.; Borsari, M.; Monte, F. D.
abstract

Cofilins are small protein of the actin depolymerizing family. Actin polymerization/depolymerization is central to a number of critical cellular physiological tasks making cofilin a key protein for several physiological functions of the cell. Cofilin activity is mainly regulated by phosphorylation on serine residue 3 making this post-translational modification key to the regulation of myofilament integrity. In fact, in this form, the protein segregates in myocardial aggregates in human idiopathic dilated cardiomyopathy. Since myofilament network is an early target of oxidative stress we investigated the molecular changes induced by oxidation on cofilin isoforms and their interplay with the protein phosphorylation state to get insight on whether/how those changes may predispose to early protein aggregation. Using different and complementary approaches we characterized the aggregation properties of cofilin-2 and its phosphomimetic variant (S3D) in response to oxidative stress in silico, in vitro and on isolated cardiomyocytes. We found that the phosphorylated (inactive) form of cofilin-2 is mechanistically linked to the formation of an extended network of fibrillar structures induced by oxidative stress via the formation of a disulfide bond between Cys39 and Cys80. Such phosphorylation-dependent effect is likely controlled by changes in the hydrogen bonding network involving Cys39. We found that the sulfide ion inhibits the formation of such structures. This might represent the mechanism for the protective effect of the therapeutic agent Na2S on ischemic injury.

2020 - Tuning of halobenzenes uptake in montmorillonite from gas phase through a functionalization process involving Cu(II)-phenanthroline and heptanethiol [Articolo su rivista]
Castellini, Elena; Malferrari, Daniele; Bernini, Fabrizio; Bighi, Beatrice; Mucci, Adele; Sainz Diaz, Ignacio Claro; Serrano, Aida; Castro, German R.; Brigatti, Maria Franca; Borsari, Marco
abstract

In this paper, montmorillonite functionalized with a Cu(II)-phenanthroline complex (Mt-CuPhen) and then exposed to heptanethiol vapors (Mt-CuPhen-HPT) was tested for the capture of halobenzenes in gas phase. The treatment of Mt-CuPhen with heptanethiol vapors produced important compositional changes involving Cu(II) reduction, oxidation of thiol to disulfide and dehydration of the mineral interlayer space. Entrapping experiments were carried out on fluorobenzene, chlorobenzene and bromobenzene at 25 and 45°C. The hybrid material proved to be very efficient for all halobenzenes and especially for fluorobenzene. Observed performances are slightly affected by temperature. Halobenzene desorption occurred even at 100°C, a temperature quite lower than that required for decomposition of Mt-CuPhen-HPT. The Mt-CuPhen-HPT hybrid material, therefore, can be reused several times and requires low energy costs for regeneration. An alternative regeneration procedure based on disulfide removal has been proposed as well. Mt-CuPhen-HPT was extensively characterized before and after halobenzenes immobilization through elemental analysis, UV-Vis measurements, thermogravimetric analyses, FTIR, NMR and X-ray absorption spectroscopies.

2020 - Urea-induced denaturation of immobilized yeast iso-1 cytochrome c: role of Met80 and Tyr67 in the thermodynamics of unfolding and promotion of pseudoperoxidase and nitrite reductase activities [Articolo su rivista]
Lancellotti, Lidia; Borsari, Marco; Bellei, Marzia; Bonifacio, Alois; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract

The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc ) immobilized on a decane-1-thiol coated gold electrode subjected to the denaturing action of urea were studied through variable temperature cyclic voltammetry and Surface-Enhanced Resonance Raman spectroscopy (SERRS). We found that the His/OH - axial heme iron coordination in both variants is unaffected by urea up to 7 M, although some conformational changes occur that increase exposure of the heme center to solvent. The thermodynamics of the unfolding process were determined with an unprecedented approach, which can be of general use for electroactive proteins. The free energy of unfolding for both variants includes relevant entropic contributions and is lower than that for the species carrying an intact Met/His coordination, consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Their lower conformational stability results in a different interaction with the MUA/MU SAM compared to the His/Met ycc forms. Denaturation invariably slows down the heterogeneous electron transfer process, but its effect on the activation enthalpy and pre-exponential factor differs for the species with and without His/Met axial heme ligation. In particular, urea unfolding of the M80A and M80A/Y67A mutants lowers the structural restraint to the heterogeneous ET. Here we show that removal of the Met ligand and an increased accessibility of the heme center to solvent through partial protein unfolding– which mimic the molecular stress experienced by mammalian cytochromes c upon binding to cardiolipin in the early events of apoptosis - add up to transform cytochrome c into an efficient electrocatalyst toward the reduction of hydrogen peroxide and nitrite.

2019 - Electrocatalytic Properties of Immobilized Heme Proteins: Basic Principles and Applications [Articolo su rivista]
Ranieri, Antonio; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Battistuzzi, Gianantonio; Sola, Marco; Borsari, Marco
abstract

Heme proteins encompass redox enzymes, electron transferases, and species for dioxygen transport and storage. Upon immobilization on a conductive surface, heme proteins can accomplish bioelectrocatalysis. In this process, they carry out oxidation or reduction of substrates at a solid electrode acting as electron acceptor or donor, respectively, thanks to electron transfer processes occurring at the interphase. The efficiency of bioelectrocatalysis depends on the electrical communication of the protein with the electrode surface, retention of protein structure upon adsorption and accessibility of the substrate to the active site. This Minireview outlines the main factors affecting bioelectrocatalysis by adsorbed heme proteins, highlights open issues, and summarizes recent advances in the field.

2019 - Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions [Articolo su rivista]
Olivé, Montse; Engvall, Martin; Ravenscroft, Gianina; Cabrera-Serrano, Macarena; Jiao, Hong; Bortolotti, Carlo Augusto; Pignataro, Marcello; Lambrughi, Matteo; Jiang, Haibo; Forrest, Alistair R. R.; Benseny-Cases, Núria; Hofbauer, Stefan; Obinger, Christian; Battistuzzi, Gianantonio; Bellei, Marzia; Borsari, Marco; Di Rocco, Giulia; Viola, Helena M.; Hool, Livia C.; Cladera, Josep; Lagerstedt-Robinson, Kristina; Xiang, Fengqing; Wredenberg, Anna; Miralles, Francesc; José Baiges, Juan; Malfatti, Edoardo; Romero, Norma B.; Streichenberger, Nathalie; Vial, Christophe; Claeys, Kristl G.; Straathof, Chiara S. M.; Goris, An; Freyer, Christoph; Lammens, Martin; Bassez, Guillaume; Kere, Juha; Clemente, Paula; Sejersen, Thomas; Udd, Bjarne; Vidal, Noemí; Ferrer, Isidre; Edström, Lars; Wedell, Anna; Laing, Nigel G.
abstract

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.

2019 - Organic Electrochemical Synthesis of hydrovanilloin, a new bioavailable nontoxic substitute of bisphenol A [Relazione in Atti di Convegno]
Lancellotti, Lidia; Maletti, Laura; Borsari, Marco; Roncaglia, Fabrizio; Tassi, Lorenzo
abstract

2019 - Structural properties of adsorbent phyllosilicates rule the entrapping ability of intercalated iron-phenanthroline complex towards thiols [Articolo su rivista]
Castellini, E.; Malferrari, D.; Bernini, F.; Mucci, A.; Borsari, M.; Brigatti, M. F.
abstract

The interaction of volatile organic sulfur derivatives, such as 1-heptanethiol (C7H16S), with clay minerals treated with a μ-oxo Fe3+-phenanthroline 1:1 complex results strongly affected by crystal chemical properties of pristine mineral phases. In particular, two sepiolite clays with different structural features demonstrated significantly different ability to immobilize the Fe3+-phenanthroline complex at two pH values (pH = 5.4 and pH = 2.3). The most effective binding was obtained with sepiolite with higher structural disorder at pH 5.4. Accordingly, the resulting hybrid material showed also the greatest efficiency in removal of thiol in gas phase. A direct correlation can be established between the adsorption of the Fe3+-phenanthroline complex and the gas binding process at room temperature. In fact, 1-heptanethiol entrapping occurs via redox reactions between Fe3+ and a first thiol molecule to give the reduced Fe2+-phenanthroline complex and disulfide, followed by the binding of further thiols to the reduced metal centre. The extremely high amount of thiol immobilized by the hybrid material also suggests the co-presence of a catalytic mechanism that guarantees the reoxidation of Fe+2 to Fe+3 and the restoration of redox reactions with thiol. Investigation and conclusions were supported by the several experimental techniques: elemental analysis, X-ray powder diffraction analyses, UV–Vis measurements, FT-IR and NMR spectroscopies, thermogravimetric analyses.

2019 - Sulfur bearing and aromatic compound trapping by layered silicates: a great start for innovative technological applications [Abstract in Atti di Convegno]
Malferrari, D.; Bernini, F.; Bighi, B.; Borsari, M.; Brigatti, M. F.; Castellini, E.; Mucci, A.; Sebastianelli, L.
abstract

2019 - Trapping at the Solid-Gas Interface: Selective Adsorption of Naphthalene by Montmorillonite Intercalated with a Fe(III)-Phenanthroline Complex [Articolo su rivista]
Castellini, E.; Malferrari, D.; Bernini, F.; Sainz Diaz, C. I.; Mucci, A.; Sola, M.; Brigatti, M. F.; Borsari, M.
abstract

In this study, stable hybrid materials (Mt-Fe(III)Phen), made by the μ-oxo Fe(III)-phenanthroline complex [(OH 2 ) 3 (Phen)FeOFe(Phen)(OH 2 ) 3 ] 4+ (Fe(III)Phen) intercalated in different amounts into montmorillonite (Mt), were used as a trap for immobilizing gaseous benzene and naphthalene and their mono chloro-derivatives at 25 and 50 °C. The entrapping process was studied through elemental analysis, magic angle spinning NMR spectroscopy, thermal analysis, and evolved gas mass spectrometry. Naphthalene and 1-chloronaphthalene were found to be immobilized in large amount at both temperatures. Molecular modeling allowed designing of the structure of the interlayer in the presence of the immobilized aromatic molecules. Adsorption is affected by the amount of the Fe complex hosted in the interlayer of the entrapping hybrid materials. On the contrary, under the same conditions, benzene and chlorobenzene were not adsorbed. Thermal desorption of naphthalenes was obtained under mild conditions, and immobilization was found to be reversible at least for 20 adsorption/desorption cycles.

2018 - Bi-allelic mutations in MYL1 cause a severe congenital myopathy [Articolo su rivista]
Ravenscroft, G.; Zaharieva, I. T.; Bortolotti, C. A.; Lambrughi, M.; Pignataro, M.; Borsari, M.; Sewry, C. A.; Phadke, R.; Haliloglu, G.; Ong, R.; Goullee, H.; Whyte, T.; Consortium, U. K.; Manzur, A.; Talim, B.; Kaya, U.; Osborn, D. P. S.; Forrest, A. R. R.; Laing, N. G.; Muntoni, F.
abstract

Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, ∼50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain (ELC) in both probands. A homozygous essential splice acceptor variant (c.479-2A > G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modelling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin ELC is critical for myofibre development and function. Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with severe congenital myopathy.

2018 - Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations [Articolo su rivista]
Bertini, Luca; Breglia, Raffaella; Lambrughi, Matteo; Fantucci, Piercarlo; De Gioia, Luca; Borsari, Marco; Sola, Marco; Bortolotti, Carlo Augusto; Bruschi, Maurizio
abstract

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

2018 - Chemical trapping of gaseous H⁠2S at high and low partial pressures by an iron complex immobilized inside the montmorillonite interlayer [Articolo su rivista]
Malferrari, Daniele; Castellini, Elena; Bernini, Fabrizio; Serrano Rubio⁠, Aida; Rafael Castro, German; Ignacio Sainz-Díaz⁠, Claro; Caleffi, Matteo; Brigatti, Maria Franca; Borsari, Marco
abstract

A stable hybrid material (Mt-Fe(III)Phen) formed by intercalation of the μ-oxo Fe(III)-phenanthroline complex [(OH⁠2)⁠3(Phen)FeOFe(Phen)(OH⁠2)⁠3]⁠4+ (Fe(III)Phen) in montmorillonite (Mt) is able to immobilize H⁠2S in gaseous phase with high efficiency even at extremely low pressures. DR UV–vis and I.R. spectroscopies, elemental analysis, X-ray powder diffraction, thermal analysis coupled with evolved gas mass spectrometry, and X-ray absorption spectroscopy show that the material has high adsorption capacity, performs fast H⁠2S trapping and is long-lasting. Moreover, even extremely low levels of H⁠2S can be removed easily and quickly from gaseous phase using a suitable amount of the trapping material. The immobilization mechanism likely involves a redox reaction between iron (III) and one S⁠2− ion, followed by the binding of a second S⁠2− ion to the metal centre. The process takes place at room temperature, is reversible for several cycles, and does not require pre-treatment of neither gaseous H⁠2S nor the adsorbent material. Therefore, this modified montmorillonite is a promising material to get rid of H⁠2S in processes of environmental interest and to obtain gaseous (and gasifiable) high quality hydrocarbons in fuels refineries.

2018 - Experimental and Theoretical Investigation of Intercalation and Molecular Structure of Organo-Iron Complexes in Montmorillonite [Articolo su rivista]
Sainz-Díaz, C. I.; Bernini, F.; Castellini, E.; Malferrari, D.; Borsari, M.; Mucci, A.; Brigatti, M. F.
abstract

The intercalation of the μ-oxo Fe(III)-phenanthroline 1:1 complex [(OH2)3(Phen)FeOFe(Phen)(OH2)3]+4 inside montmorillonite yielded a nanostructured material with strong and selective entrapping ability toward thiol molecules and hydrogen sulfide. In this work, experiments and computational molecular modeling by means of quantum mechanical calculations has been applied to study the molecular structure and interactions between this complex and the interlayer of montmorillonite. This approach allowed the identification of the geometrical disposition of the complexes inside the interlayer, the characterization of the hydration and coordination water molecules, and the explanation of the physico-chemical properties of these functionalized materials. The antiferromagnetic spin configuration of the Fe(III) ions results in the most stable state. Two conformers of the complex have been considered, having the phenanthroline rings in twisted or in parallel planes, respectively, and the transition of one conformer into the other has been explored by molecular dynamics simulations. The conformer with phenanthroline rings in parallel planes is found to be the favored species for intercalation in montmorillonite. Both experimental nuclear magnetic resonance analysis and adsorption isotherms are consistent with the modeling results. Different complex amount, equal and double of the cation exchange capacity (CEC) of montmorillonite, and hydration states inside the interlayer have been investigated reproducing faithfully the experimental d(001) spacing of the montmorillonite in the different conditions. The complex molecules intercalated over the CEC of montmorillonite adopt a disposition of the phenanthroline rings perpendicular to that of the complex already introduced by cation exchange.

2018 - Filling the Gap in Extended Metal Atom Chains: Ferromagnetic Interactions in a Tetrairon(II) String Supported by Oligo-α-pyridylamido Ligands [Articolo su rivista]
Nicolini, Alessio; Galavotti, Rita; Barra, Anne-Laure; Borsari, Marco; Caleffi, Matteo; Luo, Guangpu; Novitchi, Ghenadie; Park, Kyungwha; Ranieri, Antonio; Rigamonti, Luca; Roncaglia, Fabrizio; Train, Cyrille; Cornia, Andrea
abstract

The stringlike complex [Fe4(tpda)3Cl2] (2; H2tpda = N2,N6-bis(pyridin-2-yl)pyridine-2,6-diamine) was obtained as the first homometallic extended metal atom chain based on iron(II) and oligo-α-pyridylamido ligands. The synthesis was performed under strictly anaerobic and anhydrous conditions using dimesityliron, [Fe2(Mes)4] (1; HMes = mesitylene), as both an iron source and a deprotonating agent for H2tpda. The four lined-up iron(II) ions in the structure of 2 (Fe⋯Fe = 2.94-2.99 Å, Fe⋯Fe⋯Fe = 171.7-168.8°) are wrapped by three doubly deprotonated twisted ligands, and the chain is capped at its termini by two chloride ions. The spectroscopic and electronic properties of 2 were investigated in dichloromethane by UV-vis-NIR absorption spectroscopy, 1H NMR spectroscopy, and cyclic voltammetry. The electrochemical measurements showed four fully resolved, quasi-reversible one-electron-redox processes, implying that 2 can adopt five oxidation states in a potential window of only 0.8 V. Direct current (dc) magnetic measurements indicate dominant ferromagnetic coupling at room temperature, although the ground state is only weakly magnetic. On the basis of density functional theory and angular overlap model calculations, this magnetic behavior was explained as being due to two pairs of ferromagnetically coupled iron(II) ions (J = -21 cm-1 using JŜi·Ŝj convention) weakly antiferromagnetically coupled with each other. Alternating-current susceptibility data in the presence of a 2 kOe dc field and at frequencies up to 1.5 kHz revealed the onset of slow magnetic relaxation below 2.8 K, with the estimated energy barrier Ueff/kB = 10.1(1.3) K.

2018 - The influence of the Cys46/Cys55 disulfide bond on the redox and spectroscopic properties of human neuroglobin. [Articolo su rivista]
Bellei, Marzia; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Borsari, Marco; Lancellotti, Lidia; Ranieri, Antonio; Sola, Marco; Battistuzzi, Gianantonio
abstract

Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using Uv-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the S-S bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (deltaH°’rc) and entropy (deltaS°’rc), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH 3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH 5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate.

2017 - Baseline studies of The Clay Minerals Society Source Clay montmorillonite STx-1b [Articolo su rivista]
Castellini, Elena; Malferrari, Daniele; Bernini, Fabrizio; Brigatti, Maria Franca; Castro, German Rafael; Medici, Luca; Mucci, Adele; Borsari, Marco
abstract

For more than forty years, The Clay Minerals Society has dispensed a set of source clays which have enabled a large number of researchers to work on identical materials. Many of these source clays remained unchanged over the years but, conversely, other clays have gone out of stock and thus were replaced. This was the fate of montmorillonite STx-1a, which was replaced by STx-1b. Although STx-1a and STx-1b share many basic chemical and mineralogical features, some minor differences exist that can affect behavior. A baseline characterization of the source clay STx-1b, which was the objective of this study, was, therefore, necessary to provide researchers a tool useful not only for new investigation but also to compare new results obtained on STx-1b with literature data on STx-1a. This characterization was gained using traditional and advanced methods that include: 1) chemical composition (major and trace elements); 2) cation exchange capacity determination; 3) thermal analyses coupled with evolved gas mass spectrometry; 4) quantitative mineralogical characterization using powder X-ray diffraction and Rietveld-RIR (Reference Intensity Ratio) refinement; 5) X-ray absorption spectroscopy at the Fe K-edge; 6) diffuse reflectance ultraviolet-visible and infrared spectroscopies; 7) 29Si, 27Al, and 1H magic-angle spinning nuclear magnetic resonance measurements. According to this multi-analytical approach, the montmorillonite chemical formula is [4](Si7.753 Al0.247) [6](Al3.281 Mg0.558 Fe0.136 Ti0.024 Mn0.002) [12](Ca0.341 Na0.039 K0.061) O20 (OH)4.

2017 - Computational evidence support the hypothesis of neuroglobin also acting as an electron transfer species [Articolo su rivista]
Paltrinieri, Licia; DI ROCCO, Giulia; Battistuzzi, Gianantonio; Borsari, Marco; Sola, Marco; Ranieri, Antonio; Zanetti Polzi, Laura; Daidone, Isabella; Bortolotti, Carlo Augusto
abstract

Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.

2017 - Computational investigation of the electron transfer complex between neuroglobin and cytochrome c [Articolo su rivista]
Zanetti Polzi, Laura; Battistuzzi, Gianantonio; Borsari, Marco; Pignataro, Marcello; Pltrinieri, Licia Paltrinieri; Daidone, Isabella; Bortolotti, Carlo Augusto
abstract

Neuroglobin (Ngb) was the first vertebrate nerve globin to be identified. Since then, different physiological roles have been hypothesised for this hexa-coordinated globin, but its function is far from being unambiguously assigned. In a previous work, we collected first evidences of Ngb potentially taking part to electron transfer (ET) processes in vivo, investigating the redox thermodynamics of this globin. Here, we perform a computational investigation on the complex between Ngb and its putative in vivo partner cyt c and on the ET process between the two species. The simulated structure of the complex is amenable for ET in terms of distance and relative protein orientation. Moreover, the redox-dependent stability of the predicted Ngb-cyt c adduct and the very good agreement between calculated determinants to the ET rate and those of paradigmatic metalloproteins acting as electron shuttles all support a potential role of neuroglobin as an electron transfer species.

2017 - Corrigendum: Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity [Articolo su rivista]
Bernini, F.; Malferrari, D.; Pignataro, M.; Bortolotti, C. A.; Di Rocco, G.; Lancellotti, L.; Kayed, R.; Borsari, M.; Del Monte, F.; Castellini, E.; Brigatti, M. F.
abstract

2017 - Crystal chemical characterization and computational modeling of a μ-oxo Fe(III) complex with 1,10-phenanthroline clarify its interaction and reactivity with montmorillonite [Articolo su rivista]
Brigatti, Maria Franca; Díaz, Claro Ignacio Sainz; Borsari, Marco; Bernini, Fabrizio; Castellini, Elena; Malferrari, Daniele
abstract

This work provides a systematic study of the μ-oxo-di-fac-[triaqua-(1,10-phenanthroline-κ2N,N′)-iron(III)]bis(sulfate), [(OH2)3(phen)FeOFe(phen)(OH2)3] (SO4)2 (phen = phenanthroline). Crystal structure is determined by single-crystal X-ray diffraction data and refined to R = 0.039. The crystal structure is monoclinic (Z = 2), space group P21 with unit cell dimensions a = 8.5157(3), b = 17.6434(5), c = 9.9678(3) Å, β = 90.133(2)°, V = 1497.62(8) Å3. The triaqua(1,10-phenanthroline)iron(III) parts are linked through one oxo-bridge. Both Fe(III) cations show a distorted octahedral coordination. The single-crystal data are complemented by computational chemistry modeling at quantum mechanical level, X-ray powder diffraction at room and high temperature conditions and by thermal analysis. Molecular modeling suggests that the role of the crystallization water molecules is critical to establish the intermolecular interactions for the stability of the crystal structure.

2017 - Effective and Selective Trapping of Volatile Organic Sulfur Derivatives by Montmorillonite Intercalated with a μ-oxo Fe(III)-Phenanthroline Complex [Articolo su rivista]
Bernini, Fabrizio; Castellini, Elena; Malferrari, Daniele; Castro, German Rafael; Sainz Díaz, Claro Ignacio; Brigatti, Maria Franca; Borsari, Marco
abstract

The mu-oxo Fe(III) phenatithroline Complex [(OH2)(3)(Phen)FeOFe(Phen) (OH2)(3)](+4) intercalated in montmorillonite provides a stable hybrid material. In this study, the ability and efficiency of this material to immobilize thiols in gas phase, acting as a trap at the solid-gas interface, were investigated. Aliphatic thiols containing both hydrophilic and hydrophobic end groups were chosen to test the selectivity of this gas trap. DR-UV vis, IR, elemental analysis, thermal analysis and evolved gas mass spectrometry, X-ray powder diffraction, and X-ray absorption spectroscopy techniques were employed to characterize the hybrid material before and after thiol exposure and to provide information on the entrapping process. Thiol immobilization is very large, up to 21% w/w for heptanethiol. In addition, evidence was obtained that immobilization occurs through the formation of a covalent bond between the iron of the complex and the sulfur of the thiol. This provides an immobilization process characterized by a higher stability with respect to the method's based on physi-adsorption. Thiol immobilization resulted thermally reversible at least for 20 adsorption/desorption cycles. Unlike standard desulfurization processes like hydrotreating and catalytic oxidation which work at high temperatures and pressures, the present system is able to efficiently trap thiols at room temperature and pressure, thus saving energy. Furthermore,we found that the selectivity of thiol immobilization can be tuned acting on the amount of complex intercalated in montmorillonite. In particular, montmorillonite semisaturated with the complex captures both hydrophobic and hydrophilic thiols, while the saturated montmorillonite shows a strong selectivity toward the hydrophobic molecules.

2017 - Structure and function of Aspergillus niger laccase McoG [Articolo su rivista]
Marta, Ferraroni; Adrie H., Westphal; Borsari, Marco; Juan Antonio, Tamayo Ramos; Fabrizio, Briganti; Leo H., de Graaff; Willem J. H., van Berkel
abstract

The ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2’-azino-bis(3ethylbenzothiazoline-6-sulphonic acid or N,N-dimethylp-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (ks) of wild type and variants were determined (McoG wt E°’ is +453 mV), and especially the reduced ks values of H253A and H253N show strong correlation with their low activity on phenolic compounds. In summary, our results suggest that the His253 adaptation of McoG can be beneficial for the conversion of phenolic compounds.

2016 - Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes [Articolo su rivista]
DI ROCCO, Giulia; Bernini, Fabrizio; Borsari, Marco; Martinelli, Ilaria; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Ranieri, Antonio; Caselli, Monica; Sola, Marco; Ponterini, Glauco
abstract

The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes.

2016 - Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity [Articolo su rivista]
Bernini, Fabrizio; Malferrari, Daniele; Pignataro, Marcello; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Lancellotti, Lidia; Brigatti, Maria Franca; Kayed, Rakez; Borsari, Marco; Del Monte, Federica; Castellini, Elena
abstract

The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.

2015 - Electrowetting of nitro-functionalized oligoarylene thiols self-assembled on polycrystalline gold [Articolo su rivista]
Casalini, Stefano; Berto, Marcello; Bortolotti, Carlo Augusto; Foschi, Giulia; Operamolla, Alessandra; DI LAURO, Michele; Omar, Omar Hassan; Liscio, Andrea; Pasquali, Luca; Montecchi, Monica; Farinola, Gianluca M; Borsari, Marco
abstract

Four linear terarylene molecules (i) 4-nitro-terphenyl-4″-methanethiol (NTM), (ii) 4-nitro-terphenyl-3″,5″-dimethanethiol (NTD), (iii) ([1,1';4',1″] terphenyl-3,5-diyl)methanethiol (TM), and (iv) ([1,1';4',1″] terphenyl-3,5-diyl)dimethanethiol (TD) have been synthesized and their self-assembled monolayers (SAMs) have been obtained on polycrystalline gold. NTM and NTD SAMs have been characterized by X-ray photoelectron spectroscopy, Kelvin probe measurements, electrochemistry, and contact angle measurements. The terminal nitro group (-NO2) is irreversibly reduced to hydroxylamine (-NHOH), which can be reversibly turned into nitroso group (-NO). The direct comparison between NTM/NTD and TM/TD SAMs unambiguously shows the crucial influence of the nitro group on electrowetting properties of polycrystalline Au. The higher grade of surface tension related to NHOH has been successfully exploited for basic operations of digital μ-fluidics, such as droplets motion and merging.

2015 - Human Cofilin2: Towards the Comprehension of the Molecular Mechanism [Abstract in Atti di Convegno]
DI ROCCO, Giulia; Pignataro, Marcello; Bortolotti, Carlo Augusto; Castellini, Elena; Lancellotti, Lidia; Borsari, Marco; Sola, Marco; Del Monte, F.
abstract

Cofilin is an evolutionarily highly conserved protein which belongs to the ADF/cofilin family involved in the regulation of actin-filament dynamics depolymerizing and/or severing actin filaments. Phosphorylation on serine 3 inactivates cofilin [1,2] by generation of a charge repulsion between cofilin and actin, which is thought to occur without altering the protein structure [3]. In terms of physiological functions, cofilin- 2 is the least understood member of this protein family, which is present predominantly in skeletal and cardiac muscle [4-6]. In reducing media, even phosphatidylinositol 4,5-bisphosphate-bound cofilin is active, leading to actin dynamics in the vicinity of the plasma membrane. This mechanism has been proposed to explain why dendritic cells that are able to increase the thiol pool in antigen-specific T cells enable T cell activation even under oxidative stress conditions. On the contrary, cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death [7]. In this study we present the production, the physico-chemical characterization and the modelled structure of the wt and the phosphorylated-mimicking S3D variant of the human cofilin2. The study allowed the evaluation of the structural differences between the active and the inactive protein while an electrochemical and fluorometric approach provided new data to increase in the understanding of the cofilin-action mechanism. 1. Agnew BJ, Minamide LS, Bamburg JR. J Biol Chem 1995; 270:17582–17587. 2. Moriyama K, Iida K, Yahara I. Genes Cells 1996; 1:73–86. 3. Blanchoin L, Robinson RC, Choe S, Pollard TD. J Mol Biol 2000;295:203–211. 4. Bernstein BW, Bamburg JR. Trends Cell Biol 2010;20(4):187–95. 5. Agrawal PB, Joshi M, Savic Tetal.. Hum Mol Genet. 2012 May 15; 21(10): 2341–2356 6. C. Thirion et al. (Eur. J. Biochem. 268)-2001 7. Y. Samstag, I. John, G. H. Wabnitz Immunological Reviews 256 (2013) 30-47.

2015 - Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen [Articolo su rivista]
Ranieri, Antonio; Millo, D.; Di Rocco, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Sola, Marco
abstract

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc–CL) immobilized at IMM. In this work, we have studied electrochemically cytc–CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc–CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc–CL in solution. In particular, immobilized cytc–CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/− coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH– states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc–CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.

2015 - Solvent tunes the peroxidase activity of cytochrome c immobilized on kaolinite [Articolo su rivista]
Castellini, Elena; Bernini, Fabrizio; Berto, Marcello; Borsari, Marco; Sola, Marco; Ranieri, Antonio
abstract

The adsorption process and the peroxidase activity of yeast cytochrome c (ycc) immobilized on kaolinite (Kaol) were investigated in mixed ethanol/water solutions. The protein strongly adsorbs on the surface of the clay mineral and the thermodynamic adsorption constant increases with increasing ethanol concentration. The adsorption parameters suggest that in ycc a conformational transition from molten globule to helical state occurs in solution for ethanol concentration above 20%. The peroxidase activity of ycc immobilized on Kaol increases from 0% to 20% ethanol (v/v), then it progressively decreases and almost vanishes in pure ethanol. The catalytic properties of adsorbed yccwere studied in 20 and 40% ethanol solutionswhich correspond to the molten globule and to the helical state, respectively. In both cases, catalysis adheres to theMichaelis–Menten model. Themolten globule state, which binds more weakly to kaolinite than the helical state, was found to be more catalytically active. This study is meant to identify the physicochemical factors that modulate the catalytic activity of this kaolinite-based interface of broad applicability.

2015 - Stepwise structuring of the adsorbed layer modulates the physico-chemical properties of hybrid materials from phyllosilicates interacting with the μ-oxo Fe+3-phenanthroline complex [Articolo su rivista]
Bernini, Fabrizio; Castellini, Elena; Malferrari, Daniele; Borsari, Marco; Brigatti, Maria Franca
abstract

The -oxo Fe+3-phenanthroline 1:1 complex [(OH2)3(Phen)FeOFe(Phen)(OH2)3]+4 (hereafter Fe+3Phen) was successfully immobilized on montmorillonite (Mt) and kaolinite (Kt) minerals. Adsorption data on both minerals described an adsorption isotherm of VI type and were successfully fitted using two independent Frumkin isotherms. The interaction between the complex and the minerals is strong and yields two stable hybrid materials: Kt-Fe+3Phen and Mt-Fe+3Phen. DR UV-Vis, elemental analysis, TGA-MSEGA, temperature-controlled XRPD techniques were used to characterize the structural properties of the hybrid materials. These investigations showed that the Fe+3Phen adsorption occurs stepwise via the formation of a bilayer structure. The first layer is the result of a cation exchange process involving the negative charges of the mineral while the second one probably forms through stacking interaction and/or sulphate ions bridging. XRD diffraction measurements show that in Mt the second layer formation is accompanied by a super- structuring of the interlayer that changes the thermal and chemical properties of the composite material. In particular, interesting catalytic properties are observed in Mt-Fe+3Phen samples, but they are completely suppressed with the formation of the Fe+3Phen bilayer structure.

2015 - Surface Immobilized His-tagged Azurin as a Model Interface for the Investigation of Vectorial Electron Transfer in Biological Systems [Articolo su rivista]
Casalini, Stefano; Berto, Marcello; Kovtun, Alessandro; Operamolla, Alessandra; DI ROCCO, Giulia; Facci, Paolo; Liscio, Andrea; Farinola, Gianluca M.; Borsari, Marco; Bortolotti, Carlo Augusto
abstract

A model system for the electrochemical investigation of vectorial electron transfer in biological systems was designed, assembled and characterized. Gold electrodes, functionalized with a -OCH3 terminated, aromatic self-assembled monolayer, were used as a substrate for the adsorption of variants of copper- containing, redox metalloprotein azurin. The engineered azurin bears a polyhistidine tag at its C-terminus. Thanks to the presence of the solvent exposed tag, which chelates Cu2+ ions in solution, we introduced an exogenous redox centre. The different reduction potentials of the two redox centres and their positioning with respect to the surface are such that electron transfer from the exogenous copper centre and the electrode is mediated by the native azurin active site, closely paralleling electron transfer processes in naturally occurring multicentre metalloproteins

2015 - Thermodynamics and kinetics of reduction and species conversion at a hydrophobic surface for mitochondrial cytochromes c and their cardiolipin adducts [Articolo su rivista]
Ranieri, Antonio; DI ROCCO, Giulia; Millo, Diego; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Lancellotti, Lidia; Borsari, Marco; Sola, Marco
abstract

Cytochrome c(cytc) and its adduct with cardiolipin (CL) were immobilized on a hydrophobic SAM-coated electrode surface yielding a construct which mimics the environment experienced by the complex at the inner mitochondrial membrane where it plays a role in cell apoptosis. Under these conditions, both species undergo an equilibrium between a six-coordinated His/His-ligated and a five-coordinated His/- ligated forms stable in the oxidized and in the reduced state, respectively. The thermodynamics of the oxidation-state dependent species conversion were determined by temperature-dependent diffusionless voltammetry experiments. CL binding stabilizes the immobilized reduced His/- ligated form of cytc which was found previously to catalytically reduce dioxygen. Here, this adduct is also found to show pseudoperoxidase activity, catalysing reduction of hydrogen peroxide. These effects would impart CL with an additional role in the cytc-mediated peroxidation leading to programmed cell death. Moreover, Immobilized cytc exchanges electrons more slowly upon CL binding possibly due to changes in solvent reorganization effects at the protein-SAM interface.

2014 - Cadmium: Coordination Chemistry [Voce in Dizionario o Enciclopedia]
Borsari, Marco
abstract

The coordinative behavior of cadmium(II) is typical of a soft acid. Evidence of this fact is its strong interactions with S2− and HS− groups leading to the formation of highly stable complexes. Interactions with ligands containing oxygen-donor groups are also well known. The chemistry of CdII shows versatile coordination abilities, giving structures ranging from simple complexes to 1D, 2D, and 3D polymeric architectures. These polymers exhibit interesting physical properties. CdII is able to substitute ZnII in the active site of several Zn-enzymes and to interfere with the metabolism of CaII. This is why a strong interest has been devoted in the past decade to the coordination chemistry of cadmium. The coordinative behavior of the cadmium(II) ion resembles that of mercury(II) and, in a lesser extent, of zinc(II). The main coordination numbers observed for CdII are 4, 5, and 6. Owing to the larger size, CdII assumes coordination number 6 more easily than ZnII. In CdMe2 and dihalides in gas phase, CdII only binds to two ligands, but in solid and solution phase, the coordination number is higher than 2. Coordination number 3 characterizes CdX3− (X = halide) in organic media; however, in aqueous solution, CdBr3− is pyramidal owing to the binding of one or more water molecules. Coordination numbers higher than 3 are observed in solid MCdX3. In aqueous solution, CdX42− anions are tetracoordinated. Most CdX2L2 complexes show a coordination number higher than 4, but a distorted tetrahedral geometry is found in CdX2L2 when L is a P- or S-donor. Cd2+ is found to be five coordinated in CdCl53−. A very large number of Cd compounds are six coordinated, for example, [Cd(H2O)6]2+, [Cd(acac)2], and [Cd(acac)3]− (acac = acetylacetonate) have a CdO6 core. Six-coordinated CdN6 ions are formed with mono- and bidentate ligands such as imidazole or ethane-1,2-diamine. In the solid state, CdX2 compounds are six coordinated. Halide adducts of the type CdX4L2 are common; halogen bridging leads to an approximately octahedral CdII for Cl− and Br−, but I− gives either four and five-coordinated CdII. A few seven-coordinated compounds are known: in the solid state, each CdII ion in [Cd(H2O)(CH3OC6H4COO)2]n is seven coordinated in a distorted pentagonal bipyramidal geometry. Eight coordination is rare. CdF2 contains a CdF8 core in a fluorite structure. In Cd(NO)3·4H2O, the four water molecules are square planar and bidentate NO3− anions lie above and below this plane.

2014 - Cadmium: Inorganic Chemistry [Voce in Dizionario o Enciclopedia]
Borsari, Marco
abstract

This article provides an overview on the inorganic chemistry of cadmium. Cadmium is a bluish-white metal, the 67th most abundant element in the Earth's crust, and found in some minerals. The structure of the stable form at room temperature ( form) is hexagonal. Occurrence, production, and applications have been surveyed. CdII forms relatively few minerals: the most important are two sulfides, greenockite, and hawleyite. CdII, however, gives isomorphous replacement in several Zn minerals. For this reason, sphalerite (ZnS) is the main industrial source of cadmium. The metal is mainly obtained as a by-product of the metallurgy of Zn. Cadmium and its compounds find applications in several industrial materials: electrode materials in Ni–Cd batteries and solar cells; pigments in ceramics, glasses, paper, plastics, artist colors; coatings on steel, aluminum, and other nonferrous metals; specialized alloys; polymer stabilizers, and in nuclear industry and lab applications. 111Cd and 113Cd NMR spectroscopy can be used to study complexes, both in solid state and in solution, mostly to investigate the binding site of the metal ion in different metalloproteins. Toxicological and environmental risks are associated with its use. Cadmium compounds are highly toxic, and human exposure to this metal is known to be involved in cancer and other diseases. The targets are the body's cardiovascular, renal, gastrointestinal, neurological, reproductive, and respiratory systems. Cadmium does not seem to play any important role in higher organisms. The usual oxidation state of cadmium is 2+ and its chemistry closely resembles that of zinc and, to a lesser extend, mercury. Few compounds of CdI have been prepared and identified. CdII within zeolites is easily reduced by exposure to metal vapors to form cationic clusters containing Cd0 and CdI. The versatile coordination ability of CdII allows a wide variety of structures. The main compounds, ordered by metal oxidation state and bound anion, have been examined for their properties and structural aspects, both in solution and in the solid state. Several technologies have been developed to obtain cadmium compounds as nanocrystals or nanostructures. Cadmium glasses can find applications for their nonlinear optical properties.

2014 - Effect of motional restriction on the unfolding properties of a cytochrome c featuring a His/Met-His/His ligation switch [Articolo su rivista]
Ranieri, Antonio; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Borsari, Marco; Paltrinieri, Licia; DI ROCCO, Giulia; Sola, Marco
abstract

The K72A/K73H/K79A variant of cytochrome c undergoes a reversible change from a His/Met to a His/His axial heme ligation upon urea-induced unfolding slightly below neutral pH. The unfolded form displays a dramatically lower reduction potential than the folded species along with a pseudo-peroxidase activity. We have studied electrochemically the effects of urea-induced unfolding on the protein electrostatically immobilized on an electrode surface functionalized by means of a negatively charged molecular spacer. The latter mimics the electrostatic interaction with the inner mitochondrial membrane. This behavior has been compared with the unfolding of the same species in solution. This system constitutes a model to decipher the role of the above electrostatic interaction in the unfolding of cytochrome c at physiological pH upon interaction with the membrane component phospholipid cardiolipin in the early stages of the apoptosis cascade. We found that immobilization obstacles protein unfolding due to structural constraints at the interface imposed by protein-SAM interaction.

2014 - How the Reorganization Free Energy Affects the Reduction Potential of Structurally Homologous Cytochromes [Articolo su rivista]
Isabella, Daidone; Andrea, Amadei; Francesco, Zaccanti; Borsari, Marco; Bortolotti, Carlo Augusto
abstract

Differences in the reduction potential E0 among structurally similar metalloproteins cannot always be fully explained on the basis of their 3-D structures. We investigate the molecular determinants to E0 using the mixed quantum mechanics/molecular mechanics approach named perturbed matrix method (PMM); after comparison with experimental values to assess the reliability of our calculations, we investigate the relationship between the change in free energy upon reduction ΔA0 and the reorganization energy. We find that the reduction potential of cytochromes can be regarded as the result of the sum of two terms, one being mostly dependent on the energy fluctuations within a limited range around the mean transition energy and the second being mostly dependent linearly on the difference Δλ = λred – λox of the reorganization free energies for the ox → red (λred) and for the red → ox (λox) relaxations.

2014 - Influence of the dynamic interplay between protein and solvent on the redox properties of blue copper proteins [Abstract in Rivista]
Battistuzzi, Gianantonio; Paltrinieri, Licia; Borsari, Marco; Bortolotti, Carlo Augusto; Sola, Marco; C., Dennison; Corni, Stefano
abstract

We used direct electrochemistry and MD simulations to investigate the redox reactivity of native azurin and four chimeric cupredoxins, in which the ligand-containing loop of azurin has been replaced either with that of other members of the blue copper family or with synthetic sequences featuring only Ala residues. It turns out that the dynamic interplay between protein and solvent is the key factor determining the redox properties of these hallmark ET systems. In particular, the dynamics of the small, metal-binding loop region controls the outer-sphere reorganization energy. The molecular determinants to the reduction potential were also investigated. Moreover, the dynamics between the protein scaffold and the surrounding solvent proved to be crucial in determining the pKa of the protonation of the C-terminal copper binding His in the reduced proteins.

2014 - Unambiguous Assignment of Reduction Potentials in Diheme Cytochromes [Articolo su rivista]
I., Daidone; Paltrinieri, Licia; A., Amadei; Battistuzzi, Gianantonio; Sola, Marco; Borsari, Marco; Bortolotti, Carlo Augusto
abstract

Perturbed matrix method calculations are performed on a diheme cytochrome c (DHC) protein, in order to assign previously experimentally detemined reduction potentials (E°) to their corresponding heme groups. Very good agreement between calculated values to experimental data prove that the present approach can be used as a predictive tool of redox thermodynamic properties of multicenter redox proteins in the absence of experimental data, or in synergy with state-of-the art spectroscopic and electrochemical approaches to obtain a detailed picture of electron transfer processes within these complex systems.

2013 - Axial iron coordination and spin state change in a heme c upon electrostatic protein–SAM interaction [Articolo su rivista]
DI ROCCO, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Alois, Bonifacio; Valter, Sergo; Borsari, Marco; Sola, Marco
abstract

A bacterial di-heme cytochrome c binds electrostatically to a gold electrode surface coated with a negatively charged COOH-terminated SAM adopting a sort of 'perpendicular' orientation. Cyclic voltammetry, Resonance Raman and SERRS spectroscopies indicate that the high-potential C-terminal heme center proximal to the SAM's surface undergoes an adsorption-induced swapping of one axial His ligand with a water molecule, which is probably lost in the reduced form, and a low- to high-spin transition. This coordination change for a bis-His ligated heme center upon an electrostatically-driven molecular recognition is as yet unprecedented, as well as the resulting increase in reduction potential. We discuss it in comparison with the known methionine ligand lability in monoheme cytochromes c occurring upon interaction with charged molecular patches. One possible implication of this finding in biological ET is that mobile redox partners do not behave as rigid and invariant bodies, but in the ET complex are subjected to molecular changes and structural fluctuations that affect in a complex way the thermodynamics and the kinetics of the process.

2013 - How the Dynamics of the Metal-Binding Loop Region Controls the Acid Transition in Cupredoxins [Articolo su rivista]
Paltrinieri, Licia; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco; Christopher, Dennison; Bert L., de Groot; Stefano, Corni; Bortolotti, Carlo Augusto
abstract

Many reduced cupredoxins undergo a pH-dependent structural rearrangement, triggered by protonation of the His ligand belonging to the C-terminal hydrophobic loop, usually termed the acid transition. At variance with several members of the cupredoxin family, the acid transition is not observed for azurin (AZ). We have addressed this issue by performing molecular dynamics simulations of AZ and four mutants, in which the C-terminal loop has been replaced with those of other cupredoxins or with polyalanine loops. All of the loop mutants undergo the acid transition in the pH range of 4.4−5.5. The main differences between AZ and its loop mutants are the average value of the active site solvent accessible surface area and the extent of its fluctuations with time, together with an altered structure of the water layer around the copper center. Using functional mode analysis, we found that these variations arise from changes in nonbonding interactions in the second coordination sphere of the copper center, resulting from the loop mutation. Our results strengthen the view that the dynamics at the site relevant for function and its surroundings are crucial for protein activity and for metal-containing electron transferases.

2013 - Self-Assembly of Mono- And Bidentate Oligoarylene Thiols onto Polycrystalline Au [Articolo su rivista]
Casalini, S.; Berto, M.; Leonardi, F.; Operamolla, A.; Bortolotti, Carlo Augusto; Borsari, Marco; Sun, W.; DI FELICE, Rosa; Corni, Stefano; Albonetti, C.; Hassan Omar, O.; Farinola, G. M.; Biscarini, Fabio
abstract

Four thiolated oligoarylene molecules (i) 4-methoxy-terphenyl-4″-methanethiol (MTM), (ii) 4-methoxy-terphenyl-3″,5″-dimethanethiol (MTD), (iii) 4-nitro-terphenyl-4″-methanethiol (NTM), and (iv) 4-nitro-terphenyl-3″,5″-dimethanethiol (NTD) were synthesized and self-assembled as monolayers (SAMs) on polycrystalline Au electrodes of organic field-effect transistors (OFETs). SAMs were characterized by contact angle and AC/DC electrochemical measurements, whereas atomic force microscopy was used for imaging the pentacene films grown on the coated electrodes. The electrical properties of functionalized OFETs, the electrochemical SAMs features and the morphology of pentacene films were correlated to the molecular organization of the thiolated oligoarylenes on Au, as calculated by means of the density functional theory. This multi-methodological approach allows us to associate the systematic replacement of the SAM anchoring head group (viz. methanethiol and dimethanethiol) and/or terminal tail group (viz. nitro-, -NO2, and methoxy, -OCH3) with the change of the electrical features. The dimethanethiol head group endows SAMs with higher resistive features along with higher surface tensions compared with methanethiol. Furthermore, the different number of thiolated heads affects the kinetics of Au passivation as well as the pentacene morphology. On the other hand, the nitro group confers further distinctive properties, such as the positive shift of both threshold and critical voltages of OFETs with respect to the methoxy one. The latter experimental evidence arise from its electron-withdrawing capability, which has been verified by both DFT calculations and DC electrochemical measurements.

2013 - The Active Site Loop Modulates the Reorganization Energy of Blue Copper Proteins by Controlling the Dynamic Interplay with Solvent [Articolo su rivista]
Paltrinieri, Licia; Borsari, Marco; Ranieri, Antonio; Battistuzzi, Gianantonio; S., Corni; Bortolotti, Carlo Augusto
abstract

Understanding the factors governing the rate of electron transfer processes in proteins is crucial not only to a deeper understanding of redox processes in living organisms but also for the design of efficient devices featuring biological molecules. Here, molecular dynamics simulations performed on native azurin and four chimeric cupredoxins allow for the calculation of the reorganization energy and of structure-related quantities that were used to clarify the molecular determinants to the dynamics/function relationship in blue copper proteins. We find that the dynamics of the small, metal-binding loop region controls the outer-sphere reorganization energy not only by determining the exposure of the active site to solvent but also through the modulation of the redox-dependent rearrangement of the whole protein scaffold and of the surrounding water molecules.

2013 - Voltammetry of the cytochrome c-cardiolipin complex in the immobilized state. Implications in apoptosis initiation [Abstract in Rivista]
DI ROCCO, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco
abstract

A voltammetric behavior of the complex cytochrome c -Cardiolipin adsorbed on modified gold electrodes has been described

2012 - A Bis-Histidine-Ligated Unfolded Cytochrome c Immobilized on Anionic SAM Shows Pseudo-Peroxidase Activity [Articolo su rivista]
Ranieri, Antonio; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Monari, Stefano; Sola, Marco
abstract

Urea-unfolded yeast iso-1-cytochrome c electrostatically adsorbed on a gold electrode coated with an anionic self-assembled monolayer yields a heme-mediated electrocatalytic reduction of H2O2 (pseudo-peroxidase activity). Under the same conditions, native cytochrome c is inactive. In the unfolded protein, the Met80 heme iron ligand is replaced by a histidine residue yielding a bis-His-ligated form. H2O2 electrocatalysis occurs with an efficient mechanism likely involving direct H2O2 interaction with the iron(II) center and formation of a transient ferryl group. Comparison of the catalytic activity of a few urea-unfolded single and double Lys-to-Ala variants shows that the kinetic affinity of H2O2 for the heme iron and kcat of the bis-His-ligated form are strongly affected by the geometry of protein adsorption, controlled by specific surface lysine residues.

2012 - A surface-immobilized cytochrome c variant provides a pH-controlled molecular switch [Articolo su rivista]
Bortolotti, Carlo Augusto; Paltrinieri, Licia; Monari, Stefano; Ranieri, Antonio; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco
abstract

The K72A/K73H/K79A mutant of yeast iso-1-cytochrome c immobilized on a conductive substrate reversibly interconverts between the native-like, His-Met heme-ligated form and a His-His-ligated conformer with remarkably different redox and enzymatic properties. This transition is activated by changing the pH in a narrow range around neutrality.

2012 - The Reversible Opening of Water Channels in Cytochrome c Modulates the Heme Iron Reduction Potential [Articolo su rivista]
Bortolotti, Carlo Augusto; Amadei, A.; Aschi, M.; Borsari, Marco; Corni, S.; Sola, Marco; Daidone, I.
abstract

Dynamic protein–solvent interactions are fundamental for life processes, but their investigation is still experimentally very demanding. Molecular dynamics simulations up to hundreds of nanoseconds can bring to light unexpected events even for extensively studied biomolecules. This paper reports a combined computational/experimental approach that reveals the reversible opening of two distinct fluctuating cavities in Saccharomyces cerevisiae iso-1-cytochrome c. Both channels allow water access to the heme center. By means of a mixed quantum mechanics/molecular dynamics (QM/MD) theoretical approach, the perturbed matrix method (PMM), that allows to reach long simulation times, changes in the reduction potential of the heme Fe3+/Fe2+ couple induced by the opening of each cavity are calculated. Shifts of the reduction potential upon changes in the hydration of the heme propionates are observed. These variations are relatively small but significant and could therefore represent a tool developed by cytochrome c for the solvent driven, fine-tuning of its redox functionality.

2012 - Understanding the Mechanism of Short-Range Electron TransferUsing an Immobilized Cupredoxin [Articolo su rivista]
Monari, Stefano; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; S., Yanagisawa; K., Sato; C., Li; I., Salard; D., Kostrz; Borsari, Marco; Ranieri, Antonio; C., Dennison; Sola, Marco
abstract

The hydrophobic patch of azurin (AZ)from Pseudomonas aeruginosa is an important recognitionsurface for electron transfer (ET) reactions. The influenceof changing the size of this region, by mutating the Cterminalcopper-binding loop, on the ET reactivity of AZadsorbed on gold electrodes modified with alkanethiol selfassembledmonolayers (SAMs) has been studied. Thedistance-dependence of ET kinetics measured by cyclicvoltammetry using SAMs of variable chain length,demonstrates that the activation barrier for short-rangeET is dominated by the dynamics of molecular rearrangementsaccompanying ET at the AZ-SAM interface. Theseinclude internal electric field-dependent low-amplitudeprotein motions and the reorganization of interfacial watermolecules, but not protein reorientation. Interfacialmolecular dynamics also control the kinetics of shortrangeET for electrostatically and covalently immobilizedcytochrome c. This mechanism therefore may be utilizedfor short-distance ET irrespective of the type of metalcenter, the surface electrostatic potential, and the nature ofthe protein−SAM interaction.

2012 - pH and Solvent H/D Isotope Effects on the Thermodynamics and Kinetics of Electron Transfer for Electrode-Immobilized Native and Urea-Unfolded Stellacyanin [Articolo su rivista]
Ranieri, Antonio; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Sola, Marco
abstract

The thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D2O. Immobilized STC undergoes a limited conformational change that mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pKa for the acid transition (from 4.5 to 6.3) makes this electrode-SAM-protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies the role of protein solvation as effector of the thermodynamics and kinetics of ET.

2011 - Cloning, Expression and Physico-Chemical Characterization of a New Di-Heme Cytochrome c from Shewanella baltica OS155. [Articolo su rivista]
DI ROCCO, Giulia; Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Borsari, Marco; Ferrari, Erika; Monari, Stefano; Sola, Marco
abstract

The 16 kDa di-heme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in E. coli and investigated through UV-Vis, MCD and 1H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides di-heme cytochrome c (DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three -helices and a compact overall folding. The C-terminal domain is less helical, as the corresponding domain of R. sphaeroides DHC. The two heme groups are bridged by Tyr26 in correspondence of the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pKa values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and ionization of the heme propionate-7, respectively. Reduction potentials of -0.144 and -0.257 V (vs SHE), were determined for the C- and N-terminal heme group, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and found to reproduce successfully the ionic strength dependence of E°’.

2011 - Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction [Articolo su rivista]
Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco
abstract

Unfolding turns immobilized cytochrome c into a His–His ligated form endowed with catalytic activity towards O2, which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

2011 - Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study [Articolo su rivista]
Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Tavagnacco, Claudio; Borsari, Marco
abstract

The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E°’ of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E°’ value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.

2010 - Electron Transfer Properties and Hydrogen Peroxide, Electrocatalysis of Cytochrome c Variants at Positions 67 and 80 [Articolo su rivista]
Casalini, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract

Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinatingAla residue and alteration of the hydrogen bonding network in the region nearby following substitution ofTyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electrontransfer (ET) and the heme-mediated electrocatalytic reduction of H2O2. To this end, the voltammetry of theMet80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbedon carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxideconcentration. The thermodynamic study shows that insertion of His and Ala residues in place of Tyr67results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects onthe Eo′ values at pH 7, which for single and double variants range from approximately -0.200 to -0.220 V(vs SHE). On the contrary, both double variants show much lower ET rates compared to Met80Ala, mostlikely as a consequence of a change in the ET pathways. In the present nondenaturing immobilizing conditions,and with hydrogen peroxide concentrations in the micromolar range, the variants catalyze H2O2 reduction atthe electrode, whereas wild-type cytochrome c does not. H2O2 electrocatalysis occurs with an efficientmechanism likely involving a fast catalase-like process followed by electrocatalytic reduction of the resultingdioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presenceof a general acid-base residue for H2O2 recognition and binding through H-bonding in the distal heme siteis a key requisite for the reductive turnover of this substrate.

2010 - Factors affecting the electron transfer properties of an immobilized cupredoxin [Articolo su rivista]
Monari, Stefano; Battistuzzi, Gianantonio; C., Dennison; Borsari, Marco; Ranieri, Antonio; Siwek, MICHAL JAN; Sola, Marco
abstract

The ionic strength (I) dependence of the reduction thermodynamics (E°′, ΔHrc°′, and ΔSrc°′) and the kinetics of electron transfer (ET) for Pseudomonas aeruginosa azurin (AZ) adsorbed on a gold electrode coated with alkanethiolate SAMs has been investigated between pH 4.5 and 10.5 by cyclic voltammetry. The change in the reduction thermodynamics with I (sodium perchlorate) adheres to the Debye−Hckel model and allows the charges of the two redox states of AZ to be determined at different pH values. From pH 4 to 8 the protein charges are in agreement with those calculated considering the protonation states of the noncoordinating His35 and His83 residues and highlight that a single phosphate ion binds to both redox states of AZ, most likely at Lys122. A composite, Lys-based, equilibrium occurs at higher pH values, involving the loss of five protons at pH 10.5. The reduction thermodynamics extrapolated to zero I shows that the largely buried His35 dominates the electrostatic effects on E°′ for the equilibrium at around pH 7, whereas the residues involved in the high pH effect are more solvent exposed. At pH 10.5, the ET rate constants for AZ on all investigated SAMs are lower than the corresponding values at pH 4.5, probably due to a decrease in the tunneling efficiency at the AZ−SAM interface in terms of electronic coupling. It is suggested that Lys122 plays a distinctive role in this effect.

2010 - Oscillations in Energy Metabolism [Articolo su rivista]
S., Iotti; Borsari, Marco; D., Bendahan
abstract

Organisation of mitochondrial metabolism is a quintessential example of a complex dissipative system which can display dynamic instabilities. Several findings have indicated that the conditions inducing instabilities are within the physiological range and that mild perturbations could elicit oscillations. Different mathematical models have been put forth in order to explain the genesis of oscillations in energy metabolism. One model considers mitochondria as an organised network of oscillators and indicates that communication between mitochondria involves mitochondrial reactive oxygen species (ROS) production acting as synchronisers of the energy status of the whole population of mitochondria. An alternative model proposes that extramitochondrial pH variations could lead to mitochondrial oscillations. Oscillatory phenomena in energy metabolism have also been investigated in vivo on the basis of P-31 magnetic resonance spectroscopy (MRS) measurements of phosphocreatine post-exercise recovery in human and animal skeletal muscle. The corresponding results provide experimental evidences about the role exerted by cytosolic pH on oscillations. Finally a new simple non-linear mathematical model describing the overall chemical reaction of phosphocreatine recovery predicting oscillatory recovery pattern under certain experimental conditions is presented and discussed in the light of the experimental results reported so far.

2010 - Redox and Electrocatalytic Properties of Mimochrome VI, a Synthetic Heme-Peptide Adsorbed on Gold [Articolo su rivista]
Ranieri, Antonio; Monari, Stefano; Sola, Marco; Borsari, Marco; Battistuzzi, Gianantonio; Paola, Ringhieri; Flavia, Nastri; Vincenzo, Pavone; Angelina, Lombardi
abstract

MimochromeVI (MC-VI) is a synthetic heme-peptide containing a helix-heme-helix sandwich motif designed to reproduce the catalytic activity of heme oxidases. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of the electron transfer process for MC-VI immobilized through hydrophobic interactions on a gold electrode coated with a nonpolar SAM of decane-1-thiol have been determined through cyclic voltammetry. Immobilization slightly affects the reduction potential of MC-VI, which in these conditions electrocatalytically turns over molecular oxygen. This work sets the premises for the exploitation of totally-synthetic mimochrome-modified electrode surfaces for clinical and pharmaceutical biosensing.

2010 - The impact of urea-induced unfolding on the redox process of immobilised cytochrome c [Articolo su rivista]
Monari, Stefano; Diego, Millo; Ranieri, Antonio; DI ROCCO, Giulia; Gert van der, Zwan; Cees, Gooijer; Peressini, Silvia; Claudio, Tavagnacco; Peter, Hildebrandt; Borsari, Marco
abstract

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.

2009 - Active site loop dictates the thermodynamics of reduction and ligand protonation in Cupredoxins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; C., Dennison; C., Li; Ranieri, Antonio; Sola, Marco; S., Yanagisawa
abstract

The thermodynamics of reduction and His ligand protonation have been determined for a range of loopcontractionvariants of the electron transferring type 1 copper protein azurin (AZ). For AZPC, in which thenative C-terminal loop containing the Cys, His and Met ligands has been replaced with the shorter sequencefrom plastocyanin (PC) and AZAMI, in which the even shorter amicyanin (AMI) loop has been inserted, thethermodynamics of reduction match those of the protein whose loop has been introduced which aredifferent to the values for AZ. The enthalpic contribution to His ligand protonation, which is not observed inAZ, is similar in AZAMI and AMI. The thermodynamics of this process in AZPC are more dissimilar to thosefor PC. In the case of AZAMI-F, a variant possessing the (non natural) minimal loop that can bind a type 1copper site, the reduction thermodynamics are intermediate between those of AZPC and AZAMI, whilst thethermodynamic data for His ligand protonation are very similar to those for AMI. The results for AZAMI andAZPC are primarily due to protein based enthalpic effects related to the interaction of the metal withpermanent protein dipoles from the loop, and to the decreased loop length which favors His ligandprotonation in the cuprous proteins. Entropic factors related to loop flexibility have little influence becauseof constraints imposed by metal coordination and the fact that the introduced loops pack well against theAZ scaffold. Thus, the host scaffold in general plays a minor thermodynamic role in both processes,although for AZAMI-F differences in the first and second coordination spheres influence thethermodynamics of reduction

2009 - Electrochemical Response of Cytochrome c Immobilized on Smooth and Roughened Silver and Gold Surfaces Chemically Modified with 11-Mercaptounodecanoic Acid [Articolo su rivista]
D., Millo; Ranieri, Antonio; P., Gross; H. K., Ly; Borsari, Marco; P., Hildebrandt; G. J. L., Wuite; C., Gooijer; G., van der Zwan
abstract

Cyclic voltammetry was employed to determine the formal reduction potential and heterogeneous electron-transfer rate constant of cytochrome c immobilized on three different metal substrates chemically modified with 11-mercaptoundoecanoic acid. The metal substrates include smooth gold and silver electrodes as well as nanoscopically rough silver electrodes obtained via an oxidation−reduction cycle. Electrode roughening followed a protocol typically employed to prepare surface-enhanced Raman active surfaces such that the electrochemical results can be compared with those determined by surface-enhanced resonance Raman spectroscopy of cytochrome c. The roughness of the surfaces was estimated by means of atomic force microscopy. For all systems midpoint potentials were found to be −0.068 V (vs SCE), although for rough silver electrode the midpoint potential slightly shifted in time from −0.051 V to −0.068 V within 24 h. The heterogeneous electron-transfer rate constants differ for the various metal substrates and were found to be smaller by a factor of 2.5 for the rough and smooth Ag substrates compared to Au electrodes. These findings imply that it is primarily the kind of metal rather than its surface morphology that controls the thermodynamics and kinetics of interfacial redox processes of immobilized cytochrome c. The present paper reconciles the partly conflicting results obtained by electrochemical methods, usually done on Au, and surface-enhanced resonance Raman spectroscopic techniques which are usually performed on Ag electrodes.

2009 - Heterogeneous Electron Transfer of a Two-Centered Heme Protein: Redox and Electrocatalytic Properties of Surface-Immobilized Cytochrome c4 [Articolo su rivista]
Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco
abstract

The recombinant di-heme cytochrome c4 from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature an hydroxide ion axially bound to the heme iron at the N- and C-terminal domain, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups facilitates analysis of the heterogeneous and intra-heme electron transfer for these two-centered systems in which the high- and low-potential heme are swept over in the bilobal protein framework. The voltammetric behavior of these species, which experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode, unequivocally shows that intra-heme electron transfer is activated only in the immobilized proteins, as proposed previously for the homologous species from Pseudomonas stutzeri. T-dependent kinetic measurements show that for both proteins the C-lobe faces the HOOC-terminated SAM-coated electrode at a distance of slightly more than 7 Å. The reduction thermodynamics for the native and mutated heme (measured for the first time for a di-heme cytochrome c) in the diffusing regime reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. Protein-electrode orientation and efficient intra-heme ET enable the His,OH--ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes an unprecedented two-centered heme-base biocatalytic interface to be exploited for “third-generation” amperometric biosensing.

2009 - Redox Thermodynamics of cytochrome c subjected to urea induced unfolding [Articolo su rivista]
Monari, Stefano; Ranieri, Antonio; DI ROCCO, Giulia; G., van der Zwan; S., Peressini; C., Tavagnacco; D., Millo; Borsari, Marco
abstract

The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three protein forms which were assigned to a low-temperature and a high-temperature His-Met intermediate species and a bis-histidinate form (although the presence of a His-Lys form cannot be excluded). The much lower E°’ value of the bis-histidinate conformer as compared to His-Met ligated species is largely determined by the enthalpic contribution induced by axial ligand substitution. The biphasic E°’ vs. T profile for the His-Met species is due to a difference in reduction entropy between the conformers at low and high temperatures. Enthalpy-entropy compensation phenomena for the reduction reaction at varying urea concentration for all the forms of the investigated cytochromes c were addressed and discussed

2009 - Thermodynamics and kinetics of the electron transfer process of spinach plastocyanin adsorbed on a modified gold electrode [Articolo su rivista]
Ranieri, Antonio; Battistuzzi, Gianantonio; Borsari, Marco; Casalini, Stefano; Fontanesi, Claudio; Monari, Stefano; Siwek, MICHAL JAN; Sola, Marco
abstract

The reduction thermodynamics (DH0 rc and DS0 rc) and the kinetics of electron transfer for spinach plastocyaninadsorbed on a polycrystalline gold electrode coated with a mixed SAM made of 11-mercapto-1-undecanol and 11-mercapto-1-undecanoic acid were determined through cyclic voltammetry. Theadsorbed protein experiences a marked enthalpic stabilization of the oxidized state, likely due to the electrostaticinteraction of surface lysine(s) with the negatively charged SAM. The kinetic data indicate thatthe electron transfer process occurs through a tunnelling mechanism and that the distance between theprotein and the electrode surface can be calculated by the Marcus equation. The ionic strength of thesolution remarkably affects both the thermodynamics and the kinetics of the electron transfer processin a fashion which, for the former parameters, adheres to the Debye–Hückel model.

2008 - Catalytic Reduction of Dioxygen and Nitrite Ion at a Met80Ala Cytochrome c-Functionalized Electrode [Articolo su rivista]
Casalini, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

The Met80Ala variant of yeast iso-1-cytochrome c, immobilized on a gold electrode, is found toexchange electrons efficiently with it in nondenaturing conditions and to provide robust and persistent catalyticcurrents for O2 and nitrite ion reduction from pH 3 to 11. Direct covalent protein linkage to gold yields thebest electrochemical and electrocatalytic performances without drastically affecting the structural propertiesof the bound protein compared to the freely diffusing species. Therefore, this biocatalytic interface can beof use for the amperometric detection of the above species, which are of great environmental, industrial,and clinical interest, with particular reference to the exploitation in nanostructured biosensing devices. Thiswork shows that the use of a small engineered electron transfer (ET) protein, featuring an axial heme ironcoordination position available for the binding of exogenous ligands, in place of a large heme enzyme isa viable strategy for the improvement of the heterogeneous ET rate and the stability and efficiency ofsensing gold-protein interfaces over a wide range of T and pH.

2008 - Cloning, expression and physico-chemical characterization of a di-heme cytochrome c4 from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 [Articolo su rivista]
DI ROCCO, Giulia; Battistuzzi, Gianantonio; Borsari, Marco; DE RIENZO, Francesca; Ranieri, Antonio; M. L., Tutino; Sola, Marco
abstract

The 20-kDa di-heme cytochrome c4 from thepsycrophilic bacterium Pseudoalteromonas haloplanktisTAC 125 was cloned and expressed in Escherichia coli andinvestigated through UV–vis and 1H NMR spectroscopiesand protein voltammetry. The model structure was computedusing the X-ray structure of Pseudomonas stutzericytochrome c4 as a template. The protein shows unprecedentedproperties within the cytochrome c4 family,including (1) an almost nonpolar surface charge distribution,(2) the absence of high-spin heme Fe(III) states,indicative of a thermodynamically stable and kineticallyinert axial heme His,Met coordination, and (3) identical E0values for the two heme centers (+0.322 V vs the standardhydrogen elecrode). At pH extremes, both heme groupsundergo the ‘‘acid’’ and ‘‘alkaline’’ conformational transitionstypical of class I cytochromes c, involving ligandexchangeequilibria, whereas at intermediate pH valuestheir electronic properties are sensitive to several residueionizations.

2008 - Electron Transfer and Electrocatalytic Properties of Covalently Immobilized Laccases [Abstract in Atti di Convegno]
Siwek, M. J.; Borsari, Marco; Battistuzzi, Gianantonio; Monari, Stefano; Ranieri, Antonio; Sola, Marco
abstract

Electrochemical studies of covalently immobilized laccases have been performed. The electron transfer (ET) of a small laccase (SLAC) on a SAM-coated electrode was investigated. Scan rate and temperature dependent measurements were exploited to calculate the kinetic and thermodynamic parameter of heterogenus ET. SLAC and fungal laccase were both able to yield reductive electrocatalysis of nitrite and hydrogen peroxide.

2008 - Electron Transfer and Electrocatalytic Properties of the Immobilized Methionine80Alanine Cytochrome c Variant [Articolo su rivista]
Casalini, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; Ranieri, Antonio; Sola, Marco
abstract

The M80A variant of yeast iso-1-cytochrome c (cytc), which features a non-coordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM), and investigated by cyclic voltammetry at varying conditions of temperature, pH and O2 concentration. The E°’ value of M80A cytc on both SAMs is of approximately -200 mV (vs. SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous ET are dominated by the presence of an hydroxide ion as sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is the main responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and sets the premises for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.

2008 - Thermodynamic and Kinetic Aspects of the Electron Transfer Reaction of Bovine Cytochrome c Immobilized on 4-Mercaptopyridine and 11-Mercapto-1-Undecanoic Acid Film [Articolo su rivista]
Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; D., Millo; C., Gooijer; G., van der Zwan; Ranieri, Antonio; Sola, Marco
abstract

Bovine cytochrome c (cyt c) was adsorbed on apolycrystalline gold electrode coated with 4-mercaptopyridineand 11-mercapto-1-undecanoic acid self-assembledmonolayers (SAMs) and the thermodynamics and kineticsof the heterogeneous protein-electrode electron transfer(ET) reaction were determined by cyclic voltammetry. TheE0 values for the immobilized protein were found to belower than those for the corresponding diffusing species.The thermodynamic parameters for protein reduction (DH0 rcand DS0 rc) indicate that the stabilization of the ferric statedue to protein–SAM interaction is enthalpic in origin. Thekinetic data suggest that a tunneling mechanism is involvedin the ET reaction: the distance between the redox center ofthe protein and the electrode surface can be efficientlyevaluated using the Marcus equation.

2007 - Effects of mutational (Lys to Ala) surface charge changes on the redox properties of electrode-immobilized cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surfacelysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, andthe thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction weredetermined by voltammetry. The reaction thermodynamics were also measured for the same species freelydiffusing in solution. The selected lysine residues surround the heme group and contribute to the positivelycharged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coatedsurfaces. The E°¢ (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for thecorresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM.For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significantchanges in the E°¢ values, which do not simply follow the Coulomb law. The results suggest that the speciesdependentorientation of the protein (and thereby of the heme group) toward the negatively charged SAMinfluences the electrostatic interaction and the resulting E°¢ change. Moreover, these charge suppressionsmoderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donoracceptordistance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.

2007 - Exploiting immobilized engineered cytochrome c in bioelectronic sensing devices [Abstract in Rivista]
Sola, Marco; Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Casalini, Stefano
abstract

In recent years, redox metalloproteins have increasingly beenperceived as good candidates to serve as basic functional units ofnano-structured biomolecular surfaces at the heart ofinorganic/biological electronic devices such as biosensors andbiotransistors. Our approach was to engineer theheme-containing cytochrome c, which withstands extremeconditions in terms of pH, temperature and the presence ofnonaqueous solvents, turning it into a chimeric peroxidase. Wefound that the Met80Ala cytc variant immobilized on a variety of self-assembled monolayers on a gold electrode shows a remarkable ability to catalytically reduce O2 in alarge pH range (from 5 to 11.5). This behavior indicates thatengineered five-coordinate heme-containing cytcs are promisingcandidates for the amperometric reductive biosensing of molecular oxygen, also opening the way to the biosensing of hydrogen peroxide and organic hydroperoxides, of potential use to monitor the cellular oxidative stress.

2007 - Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; DE RIENZO, Francesca; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pK(a) values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pK(a) value and the corresponding experimental pK(a) value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.

2007 - Orientation-dependent kinetics of heterogeneous electron transfer for cytochrome c immobilized on gold: Electrochemical determination and theoretical prediction [Articolo su rivista]
Bortolotti, Carlo Augusto; Borsari, Marco; Sola, Marco; R., Chertkova; D., Dolgikh; A., Kotlyar; P., Facci
abstract

Abstract:A systematic comparison between electron-transfer rate constants measured electrochemically for different cysteine-bearing mutants of cytochrome c chemisorbed on gold surfaces in different orientations has been performed. Experimental data have been correlated with electronic coupling theoretical estimates obtained from two different empirical models for the kinetics of protein electron transfer, the tunneling pathway model and the average packing density model. The results indicate that both models also hold in the case of immobilized redox proteins, outlining their role in the rational design of optimized electron-transfer-based bioinorganic interfaces.

2007 - Voltammetric and surface-enhanced resonance raman spectroscopic characterization of cytochrome c adsorbed on a 4-mercaptopyridine monolayer on silver electrodes [Articolo su rivista]
D., Millo; A., Bonifacio; Gooijer, C.; Borsari, Marco; Ranieri, Antonio; G., van der Zwan
abstract

To combine voltammetric techniques with surface-enhanced resonance Raman scattering (SERRS), cytochrome c (cyt c) was immobilized on a roughened silver electrode chemically modified with a self-assembled monolayer (SAM) of 4-mercaptopyridine (PySH). All measurements were performed on the same electrode in a homemade spectroelectrochemical cell suitable for such applications. Cyt c on a PySH-SAM shows a quasi-reversible, monoelectronic, adsorption-controlled CV response with a formal reduction potential of -0.061 V (vs SCE), which is comparable to the values found for native cyt c adsorbed on different SAMs. SERRS spectra proved that cyt c adsorbed on a PySH monolayer is present in the native conformer (the B1 state). Voltammetric and SERRS experiments at high ionic strength revealed that the interaction between the SAM and the protein is electrostatic in nature. In conclusion, PySH was found to be suitable for adsorption of cyt c at SERRS-active silver surfaces. In comparison with other SAMs, PySH requires less time (10 min vs 12-18 h) to form a long-time durable and reproducible coating on the roughened electrode surface.

2007 - pH-Induced changes in adsorbed cytochrome c. Voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes [Articolo su rivista]
D., Millo; A., Bonifacio; Borsari, Marco; Gooijer, C.; Ranieri, Antonio; G., VAN DER ZWAN
abstract

Abstract:The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface. --------------------------------------------------------------------------------

2006 - A poly(alkylsulfanyl)thiophene functionalized with carboxylic groups [Articolo su rivista]
Cagnoli, Rita; Mucci, Adele; Parenti, Francesca; Schenetti, Luisa; Borsari, Marco; Lodi, Andrea; Ponterini, Glauco
abstract

Different routes, based on the Stille coupling, to the obtainment of a polythiophene bearing a carboxyhexylsulfanyl chain every two thiophene rings (PTCOOH) are here reported and discussed. Two PTCOOHs with different chain lengths were obtained: the shorter by hydrolysis of a polymeric ester precursor and the longer by direct Stille coupling from suitable monomers. They possess similar electrochemical properties but behave differently when aggregation and fluorescence are concerned. The PTCOOH obtained by hydrolysis is found to be fluorescent in a good solvent such as THF, and its fluorescence quantum yield decreases as the extent of aggregation increases. The polymer obtained by direct Stille coupling is less fluorescent, consistently with its proneness to aggregate. The PTCOOH obtained by hydrolysis is therefore more promising in view of the build-up of solid-state devices with exploitable fluorescence properties.

2006 - The redox chemistry of the covalently immobilized native and low-pH forms of yeast iso-1-cytochrome c [Articolo su rivista]
Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Borsari, Marco; P., Facci; Ranieri, Antonio; Sola, Marco
abstract

Cyclic voltammetry experiments were carried out on native Saccharomyces cerevisiae iso-1-cytochrome c and its C102T/N62C variant immobilized on bare polycrystalline gold electrode through the S-Au bond formed by a surface cysteine. Experiments were carried out at different temperatures (5-65 degrees C) and pH values (1.5-7). The E-o' value at pH 7 (+370 mV vs SHE) is approximately 100 mV higher than that for the protein in solution. This difference is enthalpic in origin and is proposed to be the result of the electrostatic repulsion among the densely packed molecules onto the electrode surface. Two additional electrochemical waves are observed upon lowering the pH below 5 (E-o' = +182 mV) and 3 (E-o', = +71 mV), which are attributed to two conformers (referred to as intermediate and acidic, respectively) featuring an altered heme axial ligation. This is the first determination of the reduction potential for low-pH conformers of cytochrome c in the absence of denaturants. Since the native form of cytochrome c can be restored, bringing back the pH to neutrality, the possibility offered by this transition to reversibly modulate the redox potential of cytochrome c is appealing for bioelectronic applications. The immobilized C102T/N62C variant, which differs from the native protein in the orientation of the heme group with respect to the electrode, shows very similar reduction thermodynamics. For both species, the rate constant for electron transfer between the heme and the electrode increases for the acidic conformer, which is also found to act as a biocatalytic interface for dioxygen reduction.

2005 - Axial ligation and polypeptide matrix effects on the reduction potential of heme proteins probed on their cyanide adducts [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Borsari, Marco; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract

The enthalpic and entropic changes accompanying the reduction reaction of the six-coordinate cyanide adducts of cytochrome c, microperoxidase-11 and a few plant peroxidases were measured electrochemically. Once the compensating changes in reduction enthalpy and entropy due to solvent reorganization effects are factorized out, it is found that cyanide binding stabilizes enthalpically the ferriheme following the order: cyochrome c > peroxidase > microperoxidase-11. The effect is inversely correlated to the solvent accessibility of the heme. Comparison of the reduction thermodynamics for the cyanide adducts of cytochrome c and plant peroxidases with those for microperoxidase-11 and myoglobin, respectively, yielded an estimate of the consequences of protein encapsulation and of the anionic character of the proximal histidine on the reduction potential of the heme-cyanide group. Insertion of the heme-CN group into the folded peptide chain of cyt c induces an enthalpy-based decrease in E-o' of approximately 100 mV, consistent with the lower net charge of the oxidized as compared to the reduced iron center, whereas a full imidazolate character of the proximal histidine stabilizes enthalpically the ferriheme by approximately 400 mV. The latter value should be best considered as an upper limit since it also includes some solvation effects arising from the nature of the protein systems being compared.

2005 - Cadmium: Inorganic and Coordination Chemistry [Capitolo/Saggio]
Borsari, Marco
abstract

Physical properties, inorganic and coordinative chemistry of Cadmium

2005 - Electrostatic effects on the thermodynamics of protonation of reduced plastocyanin [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Ranieri, Antonio; Leonardi, Alan; Sola, Marco
abstract

The L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(I) ion of the His87 ligand have been evaluated. The mutation-induced changes in transition enthalpy cannot be explained by electrostatic considerations. The existence of enthalpy/entropy (H/S) compensation within the protein series indicates that solvent-reorganization effects control the differences in transition thermodynamics. Once these compensating contributions are factorized out, the resulting modest differences in transition enthalpies turn out to be those that can be expected on purely electrostatic grounds. Therefore, this work shows that the acid transition in cupredoxins involves a reorganization of the H-bonding network within the hydration sphere of the molecule in the proximity of the metal center that dominates the observed transition thermodynamics and masks the differences that are due to protein-based effects.

2005 - Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Gw, Canters; DI ROCCO, Giulia; E., DE WAAL; Y., Arendsen; Leonardi, Alan; Ranieri, Antonio; Sola, Marco
abstract

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic memory of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.

2005 - Modulation of the free energy of reduction in Metalloproteins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Sola, Marco
abstract

The ulytimate determinants of the reduction potential in redox metalloproteins are the enthalpy changes due to coordintaion features and the electrostatics at the site, including the polarity effects exerted by the bulk solvent. The latter follows Coulomb's law unless additional enthalpic terms, due to bond breaking/formation processes associated with structural changes, are present. Entropy changes due to reduction-induced changes in protein flexibility may be minor contributors.

2005 - Role of the solvent in the oxidative process of a Hg electrode in presence of thiopyrimidine derivatives [Articolo su rivista]
Bakari, Souad; Borsari, Marco; Cannio, Maria; Gavioli, Giovanna; Ranieri, Antonio; S., Peressini; C., Tavagnacco
abstract

The electrochemical oxidation of a Hg electrode in the presence of thiopyrimidine derivatives has been investigated in a collection of organic solvents using voltammetric and polarographic techniques. Remarkable adsorption phenomena control the electrochemical process, which in all cases gives rise to a Hg(I) complex. The Laviron adsorption model is found to describe well the polarographic adsorption wave. Some correlations between adsorption parameters and solvent properties have also been observed and discussed.

2004 - Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwald's approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the Edegrees values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.

2004 - Redox thermodynamics of cytochrome c adsorbed on mercaptoundecanol monolayer electrodes [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Cannio, Maria; Ranieri, Antonio; Volponi, G.
abstract

The redox potentials E-0´ of bovine cytochrome c adsorbed on an 11-mercapto-1-undecanol/Au self-assembled monolayer electrode were studied through direct electrochemistry as a function of the temperature in non-isothermal experiments carried out in the presence of different anions and changes of the ionic strength. The thermodynamic parameters for protein reduction (DeltaH(rc)(0´) and DeltaS(rc)(0´)) re were determined for adsorbed and solution cytochrome and the differences in E-0´ discussed in terms of the enthalpic and entropic contributions. The adsorption process seems to remove the ability of perchlorate anion to bind to the protein surface, while a certain direct interaction is still retained in the case of chloride and phosphate. A moderate increase in E-0´ of adsorbed cytochrome was measured at increasing ionic strength and discussed in the light of the opposite effect observed for solution protein.

2004 - Solvent-based deuterium isotope effects on the redox thermodynamics of cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D2O with respect to H2O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E-o' occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions.

2003 - Control of metalloprotein reduction potential: Compensation phenomena in the reduction thermodynamics of blue copper proteins [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Borsari, Marco; Gw, Canters; E., de Waal; Ljc, Jeuken; Ranieri, Antonio; Sola, Marco
abstract

The reduction thermodynamics (DeltaH(rc)(o') and DeltaS(rc)(o')) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E-o', which is mainly determined by solvent reorganization effects. Analysis of all the DeltaH(rc)(o') and DeltaS(rc)(o') values available for this protein class indicates that enthalpy -entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.

2003 - Electrochemical behavior of diphenyl disulfide and thiophenol on glassy carbon and gold electrodes in aprotic media [Articolo su rivista]
Borsari, Marco; Cannio, Maria; Gavioli, Giovanna
abstract

The investigation of the electrochemical reduction processes of C6H5SSC6H5 and C6H5SH in CH3CN using cyclic voltammetry indicates a different behavior on GC and Au electrodes. On GC surface adsorption phenomena are absent, the electrochemical reduction process is irreversible and diffusion controlled. For both the starting molecules the same species, C6H5S-, is formed upon reduction. The Edegrees values of the reduction processes were determined by convolution method and the standard free energy of the S-S bond of C6H5SSC6H5 estimated. On Au surface instead, a self-assembled monolayer of C6H5SAuads originated after the S-S or S-H bond breaking can be observed by simply dipping the electrode in solution of C6H5SSC6H5 and C6H5SH, respectively. The properties of the SAM were investigated by electrochemical reduction of the adsorbed thiolates. On Au electrode the reduction processes involve C6H5SAuads, and give rise to desorbed C6H5S-. A neutral radical is obtained by. electrochemical oxidation of thiolate anion. It reacts rapidly with the electrode surface to give the S-Au bond again.

2003 - Electrochemistry of Protein Coated Surfaces [Capitolo/Saggio]
Borsari, Marco; Cannio, Maria; Ranieri, Antonio; Bellei, Marzia; S., Bakari
abstract

The redox properties of ET proteins adsorbed on electrodes have been extensively investigated over the last decade. Protein can interact with the metal surface directly or through a self assembled monolayer directly linked to the electrode. Muche effort has been devoted to the comprehension of the molecular and structural factors that control E°' and the kinetics of the electron transfer process. This article provides an overview of the most significant advanced made in this field recently.

2003 - Enthalpy-entropy compensation phenomena in the reduction thermodynamic of electron transport metalloproteins [Poster]
Battistuzzi, Gianantonio; Borsari, Marco; DI ROCCO, Giulia; Ranieri, Antonio; Sola, Marco
abstract

Partition of the enthalpic and entropic contributions to the reduction potential of electron transport metalloproteins, achieved through electrochemical means, is helpful for the understanding of the molecular determinants of this key parameter for protein function. Reduction enthalpy, which typically dominates this species, is mainly controlled by first coordination sphere effects and the electrostatics at the interface between the metal and the protein environment and the solvent. The contributors to the smaller, yet important, entropy changes include solvent reorganization effects and changes in polypeptide chain flexibility. To extent to which solvation effects concur to determine the reduction potential of this species is difficult to measure. However, insight can be gained from analysis of enthalpy-entropy compensation phenomena in the reduction thermodynamics. Following the Grunwald’s approach for the interpretation of H/S compensation for the solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E°’ values, owing to exact compensation. Thus the reduction potentials of this species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of the reduction-induced changes in protein flexibility

2003 - Redox thermodynamics of cytochrome c in mixed water-organic solvent solutions [Articolo su rivista]
Borsari, Marco; Bellei, Marzia; Tavagnacco, C; Peressini, S; Millo, D; Costa, G.
abstract

Bovine heart cytochrome c was studied through cyclic voltammetry in mixed water-organic solvent solutions under different conditions of temperature and the thermodynamic properties DeltaS(rc)(o) and DeltaH(rc)(o) calculated by the dependence of Edegrees by temperature. The effect of the organic fraction of the solvent on the Edegrees values of the native cyt c was found to be determined mainly by the decrease in dielectric constant of the medium. Specific interactions on the protein surface do not seem to play a remarkable role. The thermodynamic properties changes induced by the organic fraction have been interpreted tentatively in terms of solvation properties of cytochrome c and structural features of the protein environment. (C) 2003 Elsevier Science B.V. All rights reserved.

2003 - Substituent effects in the reduction behaviour of thio- and oxopyrimidines in non-aqueous solvents [Articolo su rivista]
Borsari, Marco; Cannio, Maria; Dallari, D; Fontanesi, Claudio; Gavioli, Giovanna; Peressini, S; Tavagnacco, C.
abstract

The electrochemical reduction of a series of thio- and oxopyrimidine derivatives has been investigated in organic solvents on mercury electrodes. In all cases the electrochemical process gave a dimeric species as the major product. The overall reduction mechanism is the same for oxo and thio derivatives, and is found to be dependent only on the nature of the ring nitrogen substituent. A 'father-son reaction' is observed when hydrogen is bound to the ring nitrogen atom: the radical anion obtained from the first electron transfer draws out the nitrogen proton of a nonreduced molecule and this then dimerizes. In the presence of a protonating agent as well as for the N-substituted derivatives, the 'father-son reaction' is not observed. Theoretical calculations have been performed to gain insight into the proposed mechanisms: the LUMO energy and the vertical electron affinity show a linear correlation with the reduction potentials. Analysis of the theoretical parameters has allowed step-by-step determination of the electrochemical reduction process. The manner in which solvent properties influence electrochemical behaviour has been examined, and the role of the acceptor number (AN) has been discussed.

2003 - UV/VIS spectroelectrochemical investigation of catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803 [Poster]
Borsari, Marco; Bellei, Marzia; Ranieri, Antonio
abstract

Catalase-peroxidases (CatGs) are prokaryotic proteins, which belong to the class I of the superfamily of plant, fungal and bacterial heme peroxidases. This class of proteins conserves the amino acid triad His/Trp/Asp in the proximal pocket and the triad Arg/Trp/His in the distal pocket. KatGs exhibit a high catalase activity and a peroxidase activity of broad specificity [1]. KatGs are the least studied class I heme peroxidases and so far only a few data are available for elucidation their characteristics and physiological roles. We investigated the redox properties of catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803 through UV/VIS spectroelectrochemistry experiments. Redox potentiometric titrations were carried out at varying temperature in an anaerobic OTTLE (optically transparent thin-layer electrode) cell, set in a “non-isothermal” configuration. The reduction potential of the ferric/ferrous couple in Synechocystis KatG measured is approximately 200 mV more negative then that Mycobacterium tubercolosis KatG [2]. The reduction potential of Synechocystis KatG is in the range of the cytohrome c peroxidase and plant ascorbate peroxidase, which are other members of peroxidase superfamily I, whereas the reduction potential of Mycobacterium tubercolosis KatG is closer to that of Mn2+-dependent peroxidases.

2002 - Conservation of the free energy change of pH-dependent isomerizations in cytochromes c and blue copper proteins [Poster]
Battistuzzi, Gianantonio; Borsari, Marco; Canters, Gerard W.; DI ROCCO, Giulia; Leonardi, Alan; Ranieri, Antonio; Sola, Marco
abstract

The thermodynamic parameters of the conformational transitions occurring at low pH in blue copper proteins (acid transition), and at high pH in cytochromes c (alkaline transition) have been determined through direct electrochemistry experiments carried out at variable pH and temperature. The former transition involves protonation and detachment from the Cu(I) ion of one histidine ligand (1), whereas the latter leads to a conformer in which the axial methione ligand of the ferriheme is substituted by a surface lysine, the transition being triggered by an as yet unidentified deprotonating residue (2). The blue copper proteins investigated were plastocyanins, R. vernicifera stellacyanin, CBP and T. versutus amicyanin. For all species but CBP the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. It is apparent that the thermodynamic “driving force” for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the G of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, but amicyanin, for which the greater exotermicity of the transition can be ascribed to peculiar features of the active site. For cytochromes c, we have found that both transition enthalpy and entropy are remarkably species-dependent, following the order: R.pal cytc2 >> beef (horse) heart cytc > yeast iso-1 cytc. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol-1), and consequently in the apparent pKa value. Therefore, enthalpy/entropy compensation phenomena compensation are common to both transitions, and indicate that solvent reorganization effects play an important role in the thermodynamics of the pH-induced conformational changes.

2002 - Conservation of the free energy change of the alkaline isomerization in mitochondrial and bacterial cytochromes c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

The thermodynamic parameters of the alkaline transition for oxidized native yeast iso-1 cytochrome c and Rhodopseudomonas palustris cytochrome c(2) (cytc(2)) have been determined through direct electrochemistry experiments carried out at variable pH and temperature and compared to those for horse and beef heart cytochromes c. We have found that both transition enthalpy and entropy are remarkably species dependent, following the order R. palustris cytc(2) much greater thanbeef (horse) heart cytc > yeast iso-1 cytc. Considering the high homology at the heme-protein interface in the native species, this variability is likely to be mainly determined by differences in the structural and solvation properties and the relative abundance of the various alkaline conformers. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol(-1)) and consequently in the apparent pK(a) value. This compensation indicates that solvent reorganization effects play an important role in the thermodynamics of the transition. This mechanism is functional to ensure a relatively high pK(a) value for the alkaline transition, which is needed to preserve His,Met ligation to the heme iron in cytochrome c at physiological pH and temperature, hence the Edegrees value required for the biological function. (C) 2002 Elsevier Science (USA). All rights reserved.

2002 - Control of cytochrome c redox potential: Axial ligation and protein environment effects [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ja, Cowan; Ranieri, Antonio; Sola, Marco
abstract

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (Edegrees') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the Edegrees' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature Edegrees' measurements. In this way, we have found that (1) the decrease in Edegrees' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetymethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases Edegrees' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall Edegrees' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.

2002 - Curcuminoids as potential new iron-chelating agents: spectroscopic, polarographic and potentiometric study on their Fe(III) complexing ability [Articolo su rivista]
Borsari, Marco; Ferrari, Erika; Grandi, Romano; Saladini, Monica
abstract

The pK(a) values of curcumin and diacetylcurcumin are, here doubtless, determined by means of spectroscopic and potentiometric measurements, and the enolic proton is the more acidic one. The interaction of Fe3+ with curcumin and diacetylcurcumin, in water/methanol 1:1 solution, leads to the fort-nation of the complex species [FeH2CU(OH)(2)] and [FeDCU(OH)(2)] (H2CU and DCU = curcumin or diacetylcurcumin monoanion, respectively) which prevails near pH 7. At more basic condition the prevailing species are [FeH2CU(OH)(3)](-) and [FeDCU(OH)(3)](-), which prevent metal hydroxide precipitation. H-1 NMR data state that the dissociated P-diketo moiety of the ligands is involved in metal chelation. The pK(a) value of the deprotonation reaction is strongly anticipated by the metal ion, as shown by UV spectral data. The stability constants, evaluated from potentiometric data, are near to that of desferrioxamine, which is, by now, the only iron-chelating agent for clinical use. (C) 2002 Elsevier Science B.V. All rights reserved.

2002 - Deuterated solvent effects on the electrochemical properties of cytochrome c [Poster]
Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

The changes in reduction potential of cytochrome c from bovine heart were factorized into the enthalpic and entropic contributions through direct cyclic voltammetry.

2002 - Redox thermodynamics of the Fe3+/Fe2+ couple in horseradish peroxidase and its cyanide complex [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

We have determined for the first time, the thermodynamics of Fe3+ reduction for horseradish peroxidase (HRP-C), an enzyme containing a five-coordinate high-spin heme which catalyzes the oxidation of a wide variety of substrates by H2O2 or other organic peroxides and is the best known example of secretory plant heme-peroxidases. We have also measured the reduction enthalpy and entropy for the six-coordinate low-spin cyanide adduct. The E°¢ values of thesespecies at various temperatures have been obtained with a UVvis spectroelectrochemical approach.

2002 - Structural basis for the molecular properties of cytochrome c6 [Articolo su rivista]
Dikiy, A; Carpentier, W; Vandenberghe, I; Borsari, Marco; Safarov, N; Dikaya, E; Van Beeumen, J; Ciurli, S.
abstract

This is a thorough biochemical, spectroscopic, electrochemical, and structural study of a cytochrome c(6) isolated from the filamentous green alga Cladophora glomerata. The protein sequence, elucidated using chemical and mass spectrometric techniques, features 91 amino acids and the characteristic CXXCH heme-binding motif found in c-type cytochromes. The protein is monomeric in both oxidation forms, thereby putting in question a functional role for protein dimerization. Direct electrochemical measurements established, for the first time, the kinetic and thermodynamic data for the redox process in a cytochrome c(6). In particular, the quasi-reversible and diffusion-controlled redox process is accompanied by negative enthalpy and entropy changes, resulting in an E-o' value of 0.352 V at 298 K. The pH-dependent properties of the oxidized protein, detected by UV-visible, NMR, and direct cyclic voltammetry, indicate the presence of two acid-base equilibria occurring in the acidic (pK(a) = 4.5) and alkaline regions (pK(a) = 9.0). NMR and electronic spectra allowed the assignment of these equilibria to deprotonation of heme propionate-7 and to replacement of the axial methionine with another ligand, respectively. The 1.3 Angstrom resolution X-ray structure of the oxidized protein, revealing a fold typical for class I cytochromes, suggests that the conserved Lys60 replaces the axial methionine at pH >9. The heme solvent accessibility is low, and no water molecules were found in the vicinity of the axial ligands of the heme Fe. A structure-based alignment of cytochromes c(6), and the direct comparison of their structures, indicate a substantial degree of identity between the tertiary structures and suggest patches involved in protein-protein interaction. In particular, the surface electrostatic potential of cytochromes c(6) features a hydrophobic region around the heme cofactor, and a backside surface rich in negative charges.

2002 - Thermodynamics of the acid transition in blue copper proteins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; G. W., Canters; E., de Waal; Leonardi, Alan; Ranieri, Antonio; Sola, Marco
abstract

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaHdegrees'(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic driving force for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.

2001 - Axial ligand and polipeptide matrix effects on the reduction thermodynamics of cytochrome c [Abstract in Rivista]
Sola, Marco; Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio
abstract

Insight into the mechanisms of reduction potential modulation in ET metalloproteins can be gained from the factorization of the corresponding enthalpic and entropic components, measured through variable temperature cyclic voltammetry experiments. We have compared the reduction thermodynamics forcytochrome c derivatives in which the axial methionine has been replaced by different ligands (ammonia, imidazole, 2-methyl-imidazole, azide and cyanide) and those for the adducts formed by microperoxidase-11 with the same ligands,plus N-acetylmethionine, glycine and phenyalanine.

2001 - Axial ligand and polypeptide matrix effects on the electrochemical properties of cytochrome c [Poster]
Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E') of cytochrome c through direct electrochemistry experiments

2001 - Chemical-Physical characterization of the Au-C6H5SSCH3 SAM through the electrochemical study of the surface [Poster]
Borsari, Marco; Cannio, Maria; Gavioli, Giovanna; Ranieri, Antonio
abstract

The Au-C6H5SSCH3 SAM bahaviour has been studied through linear voltammetry.

2001 - Control of metalloprotein reduction potential: The role of electrostatic and solvation effects probed on plastocyanin mutants [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; Menziani, Maria Cristina; DE RIENZO, Francesca; Sola, Marco
abstract

The changes in the thermodynamics of Cu(II) reduction for spinach plastocyanin induced by point mutations altering the electrostatic potential in proximity of the copper center were determined through variable temperature direct electrochemistry experiments. In particular, the functionally important surface residues Leu12 and Gln88 were replaced with charged and polar residues, and Asn38 was substituted with Asp. The mutational variations of the reduction enthalpy and entropy were analyzed with a QSPR (quantitative structure-property relationships) approach, employing global and local theoretical descriptors defined and computed on the three-dimensional protein structure. The correlations found are informative on how electrostatic and solvation effects control the E degrees' values in this species through the combined effects on the reduction enthalpy and entropy. The changes in reduction enthalpy can be justified with electrostatic considerations. Most notably, enthalpy-entropy compensation phenomena play a significant role: the entropic effects due to the insertion of charged residues determine E degrees' changes that are invariably opposite to those induced by the concomitant enthalpic effects. Therefore, the resulting E degrees' changes are small or even opposite to those expected on simple electrostatic grounds. The mutational variation in the reduction entropy appears to be linked to the hydrogen bonding donor/acceptor character of the northern part of the protein, above the metal site, and to the electrostatic potential distribution around the copper site. Both properties influence the reduction-induced reorganization of the water molecules on the protein surface in the same region.

2001 - Coordination Chemistry of Thio- and Oxo-Pyrimidine Derivatives [Articolo su rivista]
Battistuzzi, Raffaele; Battistuzzi, Gianantonio; Borsari, Marco; Cannio, Maria
abstract

Coordinative behaviour of thio- and oxo-pyrimidine towards several metal ions are reviewed emphasizing the role of the tautomeric equilibrium. Typical bonding modes, including simple donation, chelation or bridging are discussed on the basis of spectroscopic and structural results.

2001 - Effects of specific anion-protein binding on the alkaline transition of cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ranieri, Antonio; Sola, Marco
abstract

The thermodynamic parameters of the alkaline transition of beef heart ferricytochrome c have been measured through direct electrochemistry experiments carried out at variable pH and temperature in the presence of different sulfate concentrations. Sulfate is known to bind specifically to cytochrome c in a sequential manner at two surface sites. The effects of such a specific binding reflect on the thermodynamics of the transition and can be satisfactorily interpreted within the frame of the Debye-Huckel theory with simple electrostatic considerations. In particular, the increase in the thermodynamic pK(a) values (extrapolated to I = 0) upon sulfate binding turns out to be a fully enthalpic effect which can be accounted for by considering the coulombic effects of the formation of ionic couple(s) on the protein surface. This study also shows that the apparent pK(a) values at finite ionic strength are only moderately affected by the nature of the anion in solution, and differences tend to vanish at high ionic strength.

2001 - Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Gw, Canters; E., de Waal; Loschi, Lodovica; G., Warmerdam; Sola, Marco
abstract

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E-O' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E-O' changes simply based on Coulombic or coordination chemistry arguments.

2001 - Enthalpic and entropic contributions to the mutational changes in the reduction potential of blue copper proteins [Abstract in Rivista]
Sola, Marco; Battistuzzi, Gianantonio; Borsari, Marco; G. W., Canters; E., de Waal; DE RIENZO, Francesca; Loschi, Lodovica; G., Warmerdam; Menziani, Maria Cristina
abstract

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins and spinachplastocyanin following point mutations and residue ionizations were factorized into the enthalpic and entropiccontributions through variable temperature direct electrochemistry experiments. The mutational variations of thereduction enthalpy and entropy were analyzed with a QSPR (Quantitative Structure-Property Relationships) approach,employing global and local theoretical descriptors defined and computed on the three dimensional protein structure.

2001 - Medium and temperature effects on the redox chemistry of cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Sola, Marco
abstract

Cytochromes c (cytc) are ubiquitous heme-containing metalloproteins that shuttle electrons in a variety of electron-transport chains, most often central to the production of the chemical energy necessary for cell life. The reduction potential (E degrees') of the Fe3+/2+ couple is central to the physiological role of these species in that it influences the thermodynamic and kinetic features of electron-exchange reactions with redox partners. In the last two decades, voltammetric techniques exploiting the heterogeneous electron exchange between cytc and solid electrodes have proved to be particularly valuable for the determination of E degrees' values for these species and for characterizing the mechanistic and kinetic aspects of the redox process for the various cytc conformers under a variety of solution conditions. The understanding of how, and to what extent, different molecular factors control the E degrees' value in these species has been the subject of much debate. First coordination sphere effects on the heme iron and the interactions of the heme group with the surrounding polypeptide chain and the solvent are the main factors affecting E degrees' in cytc. These interactions are sensitive to medium effects such as the pH and the nature and ionic composition of the solvent. E degrees' is also strongly affected by the temperature, This article summarizes the authors' work on the effects on the selective stabilization of the two redox states of class I cytochromes c exerted by acid-base equilibria, general ionic strength effects, specific anion binding, the presence of nonaqueous solvents, and the temperature. The temperature dependence of E degrees' allows the determination of the enthalpy and entropy changes that accompany protein reduction. These parameters have proved to be informative with regard to the interplay between first coordination sphere effects and electrostatics at the heme-protein interface, including solvent dipoles, which mainly affect the reduction enthalpy, and solvent reorganization effects and differences in protein dynamics between the two oxidation states, which control the reduction entropy instead.

2001 - Redox Thermodynamics of electron transport metalloproteins [Abstract in Rivista]
Sola, Marco; Battistuzzi, Gianantonio; Borsari, Marco
abstract

Using direct electrochemistry techniques, we tackled the problem of how and to what extent intrinsic molecularfactors and medium conditions such as the pH, ionic strength, specific anion binding, the presence of non aqueoussolvents and the temperature control the reduction potential (E °') of the metal center in ET metalloproteins. Thedistinctive feature of our approach was to focus on the enthalpy and entropy changes accompanying protein reduction.These parameters revealed to be informative on the role of first coordination sphere effects, electrostatics at the metalproteininterface, solvent reorganization effects, oxidation state dependent differences in protein dynamics andenthalpy/entropy compensation phenomena as E °' effectors in cytochromes c, Fe-S proteins and blue copper proteins.

2001 - Redox properties and acid-base equilibria of zucchini mavicyanin [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; Ranieri, Antonio; Sola, Marco; B., Mondov; A., Marchesini
abstract

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E-o' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper. (C) 2001 Elsevier Science B.V. All rights reserved.

2001 - Redox properties of cytochrome c [Articolo su rivista]
BATTISTUZZI, Gianantonio; BORSARI, Marco; SOLA, Marco
abstract

The redox properties of cytochromes (cyt) c, a ubiquitous class of heme-containing electron transport proteins, have been extensively investigated over the last two decades. The reduction potential (Edegrees') is central to the chemistry of cyt c for two main reasons. First, Edegrees` influences both the thermodynamic and kinetic aspects of the electron exchange reaction with redox partners. Second, this thermodynamic parameter is remarkably sensitive to changes in the properties of the heme and the protein matrix, and hence can be profitably used for the investigation of the solution chemistry of cyt c. This research area owes much to the exploitation of voltammetric techniques for the determination of Edegrees' for metalloproteins, which dates back to the late 1970s. Since then, much effort has been devoted to the comprehension of the molecular factors that control Edegrees' in cyt c, which include first coordination sphere effects on the heme iron, the interactions of the heme group with the surrounding polypeptide chain and the solvent, and also include medium effects related to the nature and ionic composition of the solvent, pH, the presence of potential protein ligands, and the temperature. This article provides an overview of the most significant advances made in this field recently.

2001 - S-containing molecules films: characterization and use in direct electrochemistry of redox proteins [Articolo su rivista]
Borsari, Marco; Cannio, Maria; Gavioli, Giovanna; Ranieri, Antonio
abstract

The S-containing molecules show a great affinity towards metal surfaces, particularly Au, Pt, Ag and Cu. The interaction of the metal with the solution of these molecules gives rise to the formation of an adsorbed self assembled monolayer (SAM). The preparation and structural characterization of monolayers assemblies supported on gold electrode are of great interest in the studi of the interfacial phenomena and in particular the direct electrochemistry of redox proteins.

2001 - Silybin, a new iron-chelating agent [Articolo su rivista]
Borsari, Marco; C., Gabbi; Ghelfi, Franco; Grandi, Romano; Saladini, Monica; S., Severi; F., Borella
abstract

Silybin, a natural occurring flavolignan isolated from the fruits of Silibum marianum, has been reported to exert antioxidant and free radical scavenging abilities. It was suggested to act also as an iron chelator. The complexation and protonation equilibria of the ferric complex of this compound have been studied by potentiometric, spectrophotometric and electrochemical techniques. The formation of the complex silybin-Ga(III) in anhydrous DMSO-d6 has been studied by H-1 NMR spectroscopy. Mass spectrometry and infrared spectroscopy on silybin-Fe(III) complex confirm all data obtained by H-1 NMR spectroscopy. The experimental results show that silybin binds Fe(III) even at acidic pH. Different ternary complexes were observed at increasing methoxide ion concentration and their stability constants have been calculated. The results show the possible role of silybin in relation to the chelation therapy of chronic iron overload, as occurs in the treatment of Cooley's anemia. (C) 2001 Elsevier Science B.V. All rights reserved.

2000 - A refined model for [Fe3S4](0) clusters in proteins [Articolo su rivista]
Bentrop, D; Bertini, I; BORSARI, Marco; Cosenza, G; Luchinat, C; Niikura, Y.
abstract

The Bacillus schlegelii ferredoxin was characterized by electrochemical measurements

2000 - Bond-mediated electron tunneling in ruthenium-modified high-potential iron-sulfur protein [Articolo su rivista]
Babini, E; Bertini, I; Borsari, Marco; Capozzi, F; Luchinat, C; Zhang, Xy; Moura, Glc; Kurnikov, Iv; Beratan, Dn; Ponce, A; Di Bilio, Aj; Winkler, Jr; Gray, Hb
abstract

Bond-mediated electron tunneling in ruthenium-modified high-potential iron-sulfur protein was investigated through electrochemical methods

2000 - Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha [Articolo su rivista]
Borsari, Marco; Dikaya, E; Dikiy, A; Gonchar, Mv; Maidan, Mm; Pierattelli, R; Sibirny, Aa
abstract

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential. (C) 2000 Elsevier Science B.V. All rights reserved.

2000 - Redox thermodynamics of low-potential iron-sulfur proteins [Articolo su rivista]
Battistuzzi, Gianantonio; M., D'Onofrio; Borsari, Marco; Al, Macedo; Sola, Marco; Jjg, Moura; P., Rodrigues
abstract

The enthalpy and entropy changes associated with protein reduction (DeltaH degrees'(rc), DeltaS degrees'(rc)) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins. contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i.e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main results of this work can be summarized as follows.

1999 - Coordination properties of N-p-tolylsulfonyl-L-glutamic acid toward metal(II) - Part 2. Solution study on binary and ternary 2,2 '-bipyridine containing systems [Articolo su rivista]
Borsari, Marco; Menabue, Ledi; Saladini, Monica
abstract

The binary and ternary systems 2,2'-bipyridine (bipy)-M-tsglu [M=Cu-II, Cd-II, Zn-II or Pb-II, tsglu=N-p-tolylsulfonyl-L-glutamic acid, hereafter abbreviated LH3] were investigated in aqueous solution by means of polarography, electronic spectroscopy and potentiometry, in order to identify the type, number and stability of complex species, as a function of pH and metal to ligand molar ratio. The prevailing species in the binary systems is the [ML](-) [M=Cu-II, Cd-II, (PI)-I-II] where the amino acid molecule is in the trianionic form and coordinates the metal ion through both the carboxylic groups and deprotonated sulfonamidic nitrogen. This coordination mode is also maintained in the ternary bipy containing systems. The addition of the aromatic base in the metal coordination, stabilizes the [M(bipy)L](-) species [M=Cu-II, Cd-II] with respect to the binary ones, while it enables the Zn-II ion to substitute for the sulfonamide nitrogen-bound hydrogen of tsglu. (C) 1999 Elsevier Science Ltd. All rights reserved.

1999 - Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Loschi, Lodovica; Borsari, Marco; Sola, Marco
abstract

The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50 degrees C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E degrees' observed with increasing ionic strength [Delta E degrees(IS)' = (E degrees')(I=0.1 M)-(E degrees')(I=0 M) =-0.035 V at 25 degrees C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK(a) for a residue deprotonation is the key event of this conformational change.

1999 - Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP [Articolo su rivista]
Babini, E; Borsari, Marco; Capozzi, F; Eltis, Ld; Luchinat, C.
abstract

The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, one of the cluster ligands, The reduction potential of the S79P variant was 104+/-3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins, These results suggest that the influence of amide group 79 on the reduction potential of C, vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.

1999 - Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ja, Cowan; C., Eicken; Loschi, Lodovica; Sola, Marco
abstract

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and H-1 NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E degrees') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pK(a) values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S degrees'(rc) and Delta H degrees'(rc)) for the various protein conformers [low- and high-T native (N-1 and N-2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees' values very similar to those of the mammalian proteins; (ii) the N-1 - N-2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7.5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pK(a) for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c(2).

1999 - Redox thermodynamics of blue copper proteins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; F., Righi; Sola, Marco
abstract

The thermodynamic parameters of protein reduction (Delta H degrees'(rc) and Delta S degrees'(rc)) were measured for a number of blue copper proteins including spinach plastocyanin, cucumber plastocyanin, Pseudomonas aeruginosa azurin, Rhus vernicifera stellacyanin, cucumber stellacyanin, and horseradish umecyanin through voltammetric techniques in nonisothermal experiments at neutral pH. Including previous estimates for other members of the same protein family, we discuss here the thermodynamics of the electron-exchange reaction for twelve blue copper proteins from different sources. The enthalpic term (-Delta H degrees'(rc)/F) turns out to be the dominant contribution to the reduction potential in this protein class. However, the entropic term (T Delta S degrees'(rc)/F) heavily affects E degrees', especially for the azurins. These data were analyzed in the light of the structural and dynamic information available on protein folding, geometric and electronic features of copper ligation, and solvation properties of the two redox states. It is clearly seen that the reduction enthalpy of the subfamily of the phytocyanins is less negative as compared to that of the other cupredoxins, most likely owing to a stronger axial ligation of the copper ion (which results in a nearly tetrahedral coordination geometry) and the greater exposition of the site to the solvent, which are both factors that stabilize the Cu(II) ion. The reduction entropy, which in most cases is negative, is instead apparently related to the salvation properties of the site. In addition, by analogy with class I cytochromes c, an increase in protein rigidity could also contribute to the entropy loss on reduction. Finally, it is apparent that the strategy of protein control of the reduction thermodynamics in high-potential electron-transfer metalloproteins (blue copper proteins, class I cytochromes c, HiPIPs) is the same: a dominant enthalpic term arising from ligand-binding interactions and electrostatic factors at the metal/protein interface, which strongly stabilizes the reduced state, is most often opposed by a weaker entropic term due to changes in protein dynamics and salvation properties, which disfavors protein reduction.

1999 - Sintesi e caratterizzazione di butilsulfanil sexitiofeni [Abstract in Atti di Convegno]
Antolini, Luciano; Borsari, Marco; F., Goldoni; Iarossi, Dario; Mucci, Adele; Schenetti, Luisa
abstract

E' riporata la sintesi, tramite accoppiamento di Stille e accoppiamento ossidativo con cloruro ferrico, di due sexitiofeni funzionalizzati con catene butilsulfaniliche. La regiochimica della sostituzione è stata determinata tramite 1H,13C NMR in inverse detection.

1999 - Synthesis, Structural Characterization and Electronic Properties of 3,3'''''-Bis(butylsulfanyl)-2,2':5',2'':5'',2''':5''',2'''':5'''',2'''''-sexithiophene [Articolo su rivista]
Antolini, Luciano; Borsari, Marco; F., Goldoni; Iarossi, Dario; Mucci, Adele; Schenetti, Luisa
abstract

Synthesis and NMR, UV-VIS, electrochemical, mobility and X-ray characterization of 3,3'''''-bis(butylsulfanyl)-2,2': 5',2 :5 ,2': 5',2'''': 5'''',2'''''-sexithiophene are reported. This compound combines the promising properties already observed for 3,3''',3'''''-tris(butylsulfanyl)-2,2' : 5',2 : 5:,2''' : 5''''',2'''' : 5'''',2'''''-sexithiophene with a physical state more suitable for its use in field-effect transistors. In particular, the stability of the oxidized forms, the close packing in the solid state together with the mobility and on/off ratio observed make this sexithiophene of potential interest for organic semiconductors.

1999 - Thermodynamics of the alkaline transition of cytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; A., Martinelli; Sola, Marco
abstract

The apparent equilibrium constant (K-app) Of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pK(app) values is biphasic and yields two distinct sets of Delta H-o'(AT) and Delta S-o'(AT) values below and above approximately 40 degrees C. In the low-temperature range, the process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss; The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuzzi, G., Borsari, M, Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temperature ranges. Extrapolation of the K-app values at null ionic strength allowed the determination of the thermodynamic equilibrium constants (K-a) at each temperature, hence of the true standard thermodynamic parameters of the transition. The pK(a) value at 25 degrees C was found to be 8.0, A pK(app) value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Huckel theory fits the ionic strength dependence of the pK(app) values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential. of this protein class. It is apparent that the increase in the pK(app) values with increasing ionic strength is for the most part: an entropic effect.

1998 - Effects of solvent on the redox properties of cytochrome c: cyclic voltammetry and H-1 NMR experiments in mixed water-dimethylsulfoxide solutions [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Rossi, Giorgio; Sola, Marco
abstract

Bovine heart cytochrome c (cyt c) was studied through cyclic voltammetry, H-1 NMR and circular dichroism measurements in mixed water-dimethylsulfoxide (DMSO) solutions containing up to 50% DMSO by volume, under different conditions of temperature and pH. The effect of DMSO on the reduction potential of native cyt c was found to be determined mainly by the decrease in dielectric constant of the medium. No appreciable specific DMSO-protein interactions were detected. Instead, DMSO affects to some extent the conformation of alkaline cyt c and, notably, stabilizes both redox states of this form to the detriment of the native form. In particular, DMSO lowers the pK(a) of the native to alkaline transition for oxidized cyt c and increases the electrochemical reversibility of the voltammetric wave of the alkaline form. DMSO-induced changes in the reduction entropy for native and alkaline cyt c were also determined and interpreted tentatively in terms of solvation properties of the heme and structural features of the protein environment. (C) 1998 Elsevier Science S.A. All rights reserved.

1998 - Electrochemistry of 2-thio- and 2-oxo-pyrimidines in dimethyl formamide in the presence of dioxygen [Articolo su rivista]
Tavagnacco, C; Peressini, S; Costa, G; Borsari, Marco; Battistuzzi, Raffaele
abstract

Electrocatalysis of dioxygen reduction to superoxide ion in dimethylformamide is afforded by a reversible interaction with thiol or hydroxo forms of pyrimidine which takes place only on the mercury surface electrode even in the absence of transition metal ions. The interaction is revealed by a polarographic wave and by a voltammetric peak which can be recorded only in the presence of dioxygen in the pyrimidines solution. The polarographic wave and voltammetric peak are attributed to the reduction of the oxygenated pyrimidine. In the range of concentration where the polarographic limiting currents due to the reduction of the oxygenated and unoxygenated pyrimidine are of the same order of magnitude, the stoichiometric ratio dioxygen/pyrimidine is determined to be 1. The electrode process probably involves the formation of a complex of mercury with the thiol or the hydroxo form of the pyrimidine on the electrode surface. Adsorption or coadsorption of the reactants seems to be involved.

1998 - Modulation of Bacillus pasteurii cytochrome c(553) reduction potential by structural and solution parameters [Articolo su rivista]
Benini, S; Borsari, Marco; Ciurli, S; Dikiy, A; Lamborghini, M.
abstract

Direct cyclic voltammetry and H-1 NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c(553) isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c(553) at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell: revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275-308 K; Delta S degrees'= -162.7 +/- 1.2 J mol(-1) K-1, Delta H degrees' = -53.0 +/- 0.5 kJ mol(-1), Delta G degrees' = -4.5 +/- 0.1 kJ mol(-1), E degrees' = +47.0 +/- 0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c(553). Both effects are more accentuated in the high-temperature range (308-323 K; Delta S degrees'= -294.1 +/- 8.4 J mol(-1) K-1. Delta H degrees' = -93.4+\-3.1 kJ mol(-1), Delta G degrees'= -5.8 +/- 0.6 kJ mol(-1), E degrees' = +60.3 +/- 5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-ir conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Huckel theory, yields values of E-(I=0)degrees',= -25.4 +/- 1.4 mV, z(red)=-11.3, and z(ox)= -10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7-10 and an increase at more acidic pH, allowing the calculation of pK(O) = 5.5 and pK(R) = 5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c(553). H-1 NMR spectra of the oxidized paramagnetic cytochrome c(553) indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288-340 K at pH 7 and in the pH range 4.8-10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0-0.2 M causes a slight change of the H-1 NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5,2 +/- 1.1 M-1. The pH dependence of the isotropically shifted H-1 NMR signals of the oxidized cytochrome displays at least one ionization step with pK(O)=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c(553).

1998 - Palladium(II) complexes of N-sulfonyl-asparagine and glutamine. Evidence for metal coordination of the deprotonated amide nitrogen of the side-chain [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco
abstract

The binary and ternary (bpy) palladium(II) complexes of the N-sulfonyl derivatives of asparagine and glutamine were studied by polarography and electronic spectra as a function of pH and metal-to-ligand molar ratio. Binary [Pd(L-NO)] and [Pd(L-NO)(2)](2-) complexes are formed below pH 4, in which the N-sulfonyl-aminoacids act as bidentate N,O-chelate ligands through the deprotonated sulfonamide nitrogen and the carboxylate oxygen. No other binary species are detected at higher pH values. Both ligands form the ternary complex [Pd(L-NO)(bpy)] below pH 8.5. At higher pH values, the electrochemical and spectral data indicate that the ligands undergo an acid-base equilibrium with an apparent pK(a) value of about 10, which most probably corresponds to the deprotonation and metal coordination of the amide nitrogen of the side-chain. The overall stability constants for the binary and ternary complexes were determined polarographically and discussed in comparison with those for the homologous species formed by N-sulfonyl-aminoacids with a non-coordinating side-chain.

1998 - Redox properties of the basic blue protein (plantacyanin) from spinach [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; Sola, Marco
abstract

The reduction potential of spinach plantacyanin was determined through direct electrochemistry as a function of temperature and pH. This species shows a higher reduction potential than the homologous cucumber basic protein (CBP) (E-0 = +345 mV vs. +304 mV for CBP, in 0.1 M NaCl, pH 7, T = 25 degrees C), which turns out to be primarily the result of a more negative reduction enthalpy. Like CBP, spinach plantacyanin undergoes a positive entropy change upon reduction, at variance with most cupredoxins. Both species show a low-pH increase in E-0, indicative of protonation and detachment from the Cu(I) center of a histidine ligand. However, the pK(a) value for the spinach protein is sensibly higher (5.7 vs, about 3.5 for CBP). It is concluded that the copper site differs to some extent in the two species, although the main coordination features are likely conserved. The differences likely involve solvation properties, and, possibly, protein sequence in the metal domain. (C) 1998 Elsevier Science Inc. All rights reserved.

1998 - Thermodynamics of reduction of Chromatium vinosum high-potential iron-sulfur protein and its histidine depleted H42Q mutant [Articolo su rivista]
Babini, E; Borsari, Marco; Capozzi, F.
abstract

The thermodynamic parameters for the reduction reaction of the high-potential iron-sulfur protein from Chromatium vinosum, and its H42Q mutant, have been measured by direct cyclic voltammetry using a 'non-isothermal' reference electrode. Histidine 42 is mainly responsible for the pH dependence of the reduction potential (E degrees) with pK(a), values equal to 5.9 and 6.3 for the oxidized and reduced proteins, respectively. The H42Q mutant does not present this pH dependence, although an increasing E degrees at the lowest pH values is still present. The main contribution to the difference in free energy between the protonated and the neutral His42 forms is given by the enthalpic term (Delta Delta H degrees = 7.4 kJ mol(-1)), although the entropic term is not negligible. (C) 1998 Elsevier Science S.A. All rights reserved.

1997 - Anion binding to cytochrome c(2): Implications on protein-ion interactions in class I cytochromes c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Sola, Marco
abstract

The binding of several inorganic and carboxylate anions to cytochrome C-2 from Rhodopseudomonas palustris has been investigated by monitoring the salt-induced changes in the redox potential of the heme, using an interpretative model based on the extended Debye-Huckel equation. Most anions were found to interact specifically with the protein at one or multiple sites. Binding constants to the oxidized protein in the range 10(1)-10(2) M(-1) were determined from the anion concentration dependence of the chemical shift of the isotropically shifted heme methyl resonances. For several anions the stoichiometry and strength of the binding to cytochrome c(2) were found comparable with those determined for mitochondrial cytochromes c, in spite of the limited sequence similarity (less than 40%) and the lower positive charge of the bacterial protein. These analogies were interpreted as indicative of the existence of common binding sites which are proposed to be located in the conserved lysine-rich domain around the solvent-exposed heme edge, which is also the surface area likely involved in the interaction with redox partners. The changes in E degrees due to partial neutralization of the positive charge of cytochrome c(2) due to specific anion binding were found comparable with those for the mitochondrial species.

1997 - Cyclic voltammetry and spectroelectrochemistry of cytochrome c(8) from Rubrivivax gelatinosus. Implications in photosynthetic electron transfer [Articolo su rivista]
Borsari, Marco; Benini, S; Marchesi, D; Ciurli, S.
abstract

We present here the electrochemical characterization of cytochrome c(8) from light-grown cells of the purple phototroph Rubrivivax gelatinosus. At 25 degrees C, cytochrome c(8) exhibits a quasi-reversible, diffusion-controlled, electrochemical process as observed by cyclic voltammetry (CV) using a pyrolitic graphite microelectrode (10 mM Tris . HCl buffer, 250 mM NaCl). The pH dependence of the reduction potential, investigated in the range 4.9-8.2, allows the calculation of E-m,E-7 = +294 mV, and further yields values of pK(OX) = 6.24 and pK(red) = 6.87, probably associated to the ionization of a heme propionate group in the oxidized and reduced form of cytochrome c(8), respectively. The calculated reduction potentials of completely protonated and deprotonated cytochrome c(8) are +322 and +284 mV, respectively. The equilibrium reduction potential of cytochrome c(8) was also determined at pH 8 by recording its visible absorption spectrum as a function of the applied potential using an optically transparent electrode (OTE) cell (E-m = +276 mV). These observations indicate that cytochrome c(8) might play a significant role in photosynthetic electron transport and associated proton translocation in purple bacteria.

1997 - Electrochemical behaviour of oligometallic sandwich complexes of cyclosiloxanolate ligands [Articolo su rivista]
Borsari, Marco; Gavioli, Giovanna; Zucchi, Claudia; Palyi, Gyula; R., Psaro; R., Ugo; Oi, Shchegolikhina; Aa, Zhdanov
abstract

The conductometric and voltammetric behaviour of 16 oligocyclosiloxanolate transition metal complexes in DMF is reported and discussed. The results of the conductometric measurements indicate that in DMF most of the compounds are electrolytes and the conductivity is due prevalently to the mobile (free) Na+ cation, the contribution of the large cluster anion being negligible, The cyclic voltammetric curves show only one reversible peak couple with E-p values very close to each other. These results suggest a high extent of electronic delocalisation in the cluster core of these sandwich type complexes and permit these compounds to be regarded as heteronuclear clusters of higher-valent transition metals.

1997 - Electrochemical processes involving a sulphonamide moiety [Articolo su rivista]
Borsari, Marco; Fontanesi, Claudio; Gavioli, Giovanna
abstract

The peculiar electrochemical and physico-chemical behaviour of RSO2 substituted compounds can be attributed to the electronwithdrawing effect induced by sulphur atom, in particular the acidity of the nitrogen-bound hydrogen and the presence of a characteristic electrochemical reduction mechanism. Adsorption process at the mercury-aqueous solution of benzenesulfonamide derivatives and RSO2N-protected aminoacids is investigated and explained using some calculated structural indexes. The relations among these indexes and the adsorption free energy indicate that substitutions on the amidic nitrogen greatly affect the interfacial behaviour. The comparison of the reduction process of RSO2 protected aminoacids with that of the sulphonamide derivatives in protic and aprotic solvents has shown that these aminoacids, in neutral form, can be better considered as sulfonamides and their electrochemical parameters fit structural correlations with theoretical indexes, as LUMO energy and localized charges, in the same way shown by sulfonamide derivatives. Using these correlations, the role of the RSO2 moiety on the amidic acidity and on the electron transfer process has been discussed also on the basis of the calculated differences in total energy of dissociated and undissociated species. Theoretical indexes allow to investigate the electron route in the electrochemical reduction and the role of the substituents on nitrogen atom in determining mechanism and energetics of the electrochemical process.

1997 - Redox Interconversion of [ReVO]3+  [Re(III)]3+ Centers in Octahedral 4,6-Dimethyl-Pyrimidine-2-Thiolate/Triphenylphosphine Rhenium(V) and Rhenium(III) Mixed Complexes [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Battistuzzi, Raffaele
abstract

Reaction of trans-[ReOX2(EtO)(PPh3)2] (X = CI, Br, 1) with 4,6-dimethylpyrimidine-2-(l H)-thione(pymSH) in 1:1 molar ratio in refluxing acetone, rapidly formed [ReOX2(pymS)(PPh3)] (X = C1, Br, I)compounds and PPh~ in good yields. Upon increasing the refluxing time (5-6 h), the above products react ina 1:2 molar ratio producing the paramagnetic trans-[ReX2(pymS)(PPh3)2] (X = CI, Br) species and triphenylphosphineoxide as a result of oxygen transfer from the [ReO] 3+ core to the PPh3. The trans-[ReX2(pymS)(PPh3)2] (X = CI, Br) species, in organic solvents at room temperature and in the presence of atmosphericoxygen, are easily oxidized back to [ReOX2(pymS)(PPh~)] with the concomitant formation ofO = PPh3. Theseoxidation reactions most likely proceed through the loss of a PPh, ligand on the reducing rhenium(IIl) center,which makes a coordination site available for the oxidative addition of dioxygen which produces a highlyreactive rhenium-dioxygen intermediate ([ReX2(pymS)(PPh3)(02)]). The time course of the aerial oxygenationreactions at room temperature was inferred from the rate [Kin = (1.30 + 0.05) x 10 2 h t] of disappearanceof the paramagnetic trans-[ReX2(pymS)(PPh3)2] (X = C1, Br) species, monitored by VIS-NIR and ~H NMRspectroscopy. Spectroscopic (UV-VIS-NIR, ~H and 3~p NMR), magnetic and electrochemical properties of thecomplexes are discussed.

1997 - Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; F., Francia; Sola, Marco
abstract

The reduction potentials of beef heart cytochrome c and cytochromes c(2) from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (Delta S(rc)degrees and Delta H(rc)degrees) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH-and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c(2) and the eukaryotic species can be separated into an enthalpic term (-Delta Delta H(rc)degrees/F) of +0.130 V and an entropic term (T Delta Delta S(rc)degrees/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees/temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent H-1 NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

1997 - Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; Sola, Marco
abstract

The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium, The most notable properties of CBP include a positive entropy change upon reduction, a low pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics, The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sires, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N2S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential.

1997 - Reductive electron transfer on trichloromethyl derivatives of benzene and pyridine studied by electrochemical methods [Articolo su rivista]
Borsari, Marco; Dallari, D; Fontanesi, Claudio; Gavioli, Giovanna; Iarossi, Dario; Piva, R; Taddei, Ferdinando
abstract

The electrochemical reduction of alpha,alpha,alpha-trichlorotoluene (benzotrichloride) and of the corresponding isomeric pyridine derivatives has been investigated by cyclic voltammetry and microcoulometry. Three irreversible diffusion controlled reduction waves have been observed in the voltammetry of the trichloromethyl derivatives and are found to correspond to the progressive two-electron reduction of derivatives where a chlorine atom is substituted by a hydrogen atom. The peak potentials E-p show that reduction of the pyridine derivatives is easier than that of the benzene derivatives with the same number of chlorine atoms, the more positive shift occurring for the 4-substituted compounds. The experimental findings would indicate a concerted electron transfer-bond breaking (C-Cl) mechanism for all the compounds examined, as well as for the chloromethyl and dichloromethyl derivatives formed after Cl/H substitution. Electron uptake appears to be the rate-determining step of the reductive cleavage of these molecules. The effects of the different aromatic rings and of the degree of halogen substitution of the methyl group on the reduction potentials are discussed.

1996 - A serine->cysteine ligand mutation in the high potential iron-sulfur protein from Chromatium vinosum provides insight into the electronic structure of the [4Fe-4S] cluster [Articolo su rivista]
Babini, E; Bertini, I; Borsari, Marco; Capozzi, F; Dikiy, A; Eltis, Ld; Luchinat, C.
abstract

We have succeeded in preparing a mutant of the High Potential Iron-Sulfur Protein (HiPIP) from Chromatium vinosum in which a cysteine ligand has been replaced by a serine (C77S). Proton chemical shift data and nuclear Overhauser effects indicate that structural perturbations induced by the C77S mutation are minimal in both the oxidized and reduced forms of the HiPIP. The reduction potential of C77S is 25 mV lower than that of the wild-type HiPIP (WT) (0.2 M ionic strength, pH 4.5-9.0, 25 degrees C). Assignment of the hyperfine shifted signals in the H-1 NMR spectrum of oxidized C77S revealed that the temperature dependences of the signals associated with cluster-ligating residues 46 and 77 are Curie and and-Curie type, respectively, and are thus the reverse of those in WT. Taken together, these observations indicate that the iron bound to Ser-77 is less reducible than the corresponding iron in WT. The results are consistent with a previous model of the electronic structure of oxidized HiPIP clusters, confirming the presence of an equilibrium between two species of differing valence distributions. The current results permit the extension of this model to predict the relative reduction potentials of the individual iron ions in the oxidized HiPIPs up to now investigated.

1996 - Amide group coordination to the Pb2+ ion [Articolo su rivista]
BATTISTUZZI, Gianantonio; BORSARI, Marco; MENABUE, Ledi; SALADINI, Monica; SOLA, Marco
abstract

The binary and ternary (2,2'-bipyridine) complexes of dipositive lead formed by N-carbonyl and N-sulfonyl amino acids, which are ligands containing the peptide and the sulfonamide group, respectively, were investigated in aqueous solution by NMR and differential pulse polarography, and some were also characterized crystallographically. N-Tosylglycine, N-tosyl-beta-alanine, and N-benzoylglycine behave as simple carboxylate ligands at acid pH, while around neutrality they switch to dianionic N,O-bidentate chelating ligands due to the involvement of the deprotonated amide nitrogen as an additional donor site. The same coordination behavior is maintained in the presence of 2,2'-bipyridine. The binary and ternary species formed in solution, and their stability constants were determined and compared with those of the homologous complexes of Pd2+, Cu2+, Cd2+, and Zn2+. The Pb2+ ion is the only dipositive metal which is effective in promoting peptide nitrogen deprotonation in benzoylglycine. The molecular structures of [Pb(N-tosylglycinato-N,O)(H2O)] (1), [Pb(N-benzoylglycinato-O)(2)-(H2O)(2)]. 2H(2)O (2), and [Pb(N-tosylglycinato-O)(2)(bpy)] (3) were determined by X-ray crystallography (O and N,O refer to the ligands binding as carboxylates and as N,O-chelating dianions, respectively). These compounds are all polymeric with six- to eight-coordinate metals showing distorted coordination geometries indicative of a stereochemically active metal lone pair. Polymerization is invariably determined by a bidentate chelate carboxylate group with one oxygen bridging between two metals, and in 2 and 3 it occurs through the formation of chains of Pb2O2 square-planar rings. The binding set in 1, involving a deprotonated amide nitrogen and a sulfonic oxygen, is unprecedented for the Pb2+ ion. This work provides new information on the solution and solid state chemistry of dipositive lead with ligands of biological interest, a research area that has received little attention in the past, although it is of great relevance for understanding the mechanisms of metal toxicity.

1996 - Amide nitrogen co-ordination of CoII and NiII in ternary 2,2′-bipyridine-containing systems. A solution and solid-state study [Articolo su rivista]
Borsari, M.; Menabue, L.; Saladini, M.
abstract

The ternary systems 2,2′-bipyridine (bipy)-M-amino acid [M = CoII or NiII; amino acid (H2L) = N-p-tolyl-sulfonylglycine (tsgly), -β;-alanine (ts-β-ala) or N-benzoylglycine (H2hip)] were investigated in aqueous solution by means of polarography, electronic spectroscopy and potentiometry, in order to identify the type, number and stability of complex species, as a function of pH and bipy : M : amino acid ratio. With tsgly the prevailing species are [MII(bipy)n(HL)]+ and [MII(bipy)nL] (n = 1 or 2), with pKNH for the deprotonation of sulfonamide nitrogen 7.8(2) and 7.4(2), for CoII and NiII, respectively and the crystal and molecular structure of [Co(bipy)2(tsglyNO)]·2H2O (NO indicates binding as a N,O-chelating dianion) was determined. The CoII is six-co-ordinated by four nitrogen atoms of the bipy moieties, the deprotonated sulfonamide nitrogen and one carboxylic oxygen of the tsgly dianion. On standing in air, an alkaline solution of bipy-CoII-tsgly turned from orange to wine-red in a few days, indicating the oxidation of CoII to CoIII. With ts-β-ala and H2hip metal hydrolysis prevents the formation of deprotonated complexes.

1996 - Amide nitrogen coordination of Co(II) and Ni(II) in ternary 2,2'-bipyridine-containing systems. A solution and solid state study. JOURNAL OF THE CHEMICAL SOCIETY DALTON TRANSACTIONS; p. , ISSN: 0300-9246 [Articolo su rivista]
Borsari, Marco; Menabue, Ledi; Saladini, Monica
abstract

Cobalt(II) and Nickel(II) interactions towards N-protected aminoacid were investigated both in solution and solid state and the ternary compexes with 2,2'-bipyridine. The experimental conditions (pH, metal:ligand:amine ratio) for amide nitrogen deprotonation in ternary systems were defined and the stability constant of ternary complexes were evaluated. The crystal structure of Co(II)bis-tosylglycinato 2,2'-bipyridine complex was determined.

1996 - Anion binding to mitochondrial cytochromes c studied through electrochemistry - Effects of the neutralization of surface charges on the redox potential [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Dallari, D; Lancellotti, Isabella; Sola, Marco
abstract

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion-protein interactions, using a Debye-Huckel-based model. In parallel, protein charge neutralization resulting from specific anion binding allows monitoring for surface-charge/E(o) relationships. This approach shows that a number of anions, most of which are of biological relevance, namely Cl-, HPO42-, HCO3-, NO3-, SO42-, ClO4-, citrate(3-) and oxalate(2-); bind specifically to the protein surface, often in a sequential manner as a result of the presence of multiple sites with different affinities, The binding stoichiometries of the various anions toward a given cytochrome are in general different. Chloride and phosphate appear to bind to a greater extent to both proteins as compared to the other anions. Differences in binding specificity toward the two cytochromes, although highly sequence-related, are observed for a few anions. The data are discussed comparatively in terms of electrostatic and geometric properties of the anions and by reference to the proposed location and amino acid composition of the anion binding sites, when available. Specific binding of this large set of anions bearing different charges allows the electrostatic effect on E(o) due to neutralization of net positive protein surface charge(s) to be monitored. H-1 NMR indeed indicates the absence of significant salt-induced structural perturbations, hence the above change in E(o) is predominantly electrostatic in origin. A systematic study of protein surface-charge/E(o) relationships using this approach is unprecedented. Values of 15-25 mV (extrapolated at zero ionic strength) are obtained for the decrease in E(o) due to neutralization of one positive surface charge, which are of the same order of magnitude as previous estimates obtained with either mutation or chemical modification of surface lysines. The effects of the anion-induced decrease of net positive charge on E(o) persist also at a relatively high ionic strength and add to the general effects related to the charge shielding of the protein as a whole due to the surrounding ionic atmosphere: hence the ionic strength dependence of the rate of electron transfer between cytochromes c and redox partners could also involve salt-induced changes in the driving force.

1996 - Cyclooligosiloxanolate cluster complexes of transition metals and lanthanides [Articolo su rivista]
Zucchi, Claudia; Oi, Shchegolikhina; Borsari, Marco; Cornia, Andrea; Gavioli, Giovanna; Ac, Fabretti; E., Rentschler; D., Gatteschi; R., Ugo; R., Psaro; Pozdniakova, Y. U. A.; Sv, Lindeman; A. A., Zhdanov; Palyi, Gyula
abstract

Cyclooligosiloxanolate complexes of higher valent transition metals and lanthanides were synthesized and characterized by single-crystal X-ray diffraction experiments, spectra, cyclic voltammetry, conductivity and magnetic behaviour. These complexes are sandwich-type clusters containing assemblies of 4 to 8 metals between siloxanolate ligand 'layers'. A high degree of electron delocalization in the metal containing fragment of these complexes is detected by electrochemical and magnetic measurements, The synthesis, crystal and molecular structure determined by X-ray diffraction of Na-6{[C6H5SiO2)(8)]Gd-2(4)(mu(4)-O)} is reported.

1996 - Cyclosiloxane sandwich complexes of a lanthanide metal: Na-6{[(C6H5SiO2)(8)](2)Nd-4(mu(4)-O)} [Articolo su rivista]
Oi, Shchegolikhina; Ya, Pozdniakova; Sv, Lindeman; Aa, Zhdanov; R., Psaro; R., Ugo; Gavioli, Giovanna; Battistuzzi, Raffaele; Borsari, Marco; T., Ruffer; Palyi, Gyula; Zucchi, Claudia
abstract

The oligocyclosiloxane cluster ('sandwich') complex of Nd3+, Na-6{[(C6H5SiO2)(8)]Nd-2(4)(mu(4)-O)} has been prepared and characterized by analyses, infrared spectra, single crystal X-ray diffraction, magnetic susceptibility, conductivity and cyclic voltammetry. The mu(4)-O2- anion is in a square planar coordination environment. Electrochemical studies identify the cluster core as a moiety with a delocalized electron system.

1996 - Effects of pH, ionic composition of the medium and temperature on the redox properties of electron carrier metalloproteins studied through voltammetric techniques. Cytochromes c as an example [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Sola, Marco
abstract

A review with 71 refs. The redox properties of cytochromes c are remarkably sensitive to pH, ionic strength, ionic compn. of the medium and temp. The effect of increasing concns. of a variety of anions on the redox potential of mitochondrial and bacterial species measured through voltammetric techniques indicate the presence of specific sites of interaction on the protein surface. Proton uptake and release and specific anion binding alter the protein charge and their effect on E° allow information to be obtained on electrostatic effects on the redox properties of the heme. The temp. dependence of the redox potential of cytochrome c changes with pH. At alk. pH values a T-induced structural change occurs above 40°.

1996 - The influence of a surface charge on the electronic and steric structure of a high potential iron-sulfur protein [Articolo su rivista]
Bertini, I; Borsari, Marco; Bosi, M; Eltis, Ld; Felli, Ic; Luchinat, C; Piccioli, M.
abstract

The recombinant high-potential iron-sulfur protein (HiPIP) iso-I from Ectothiorhodospira halophila has been mutated at position 68. The alpha C of Val 68 is within a 0.6-nm sphere from the closest iron ion of the cluster, The valine residue has been replaced by a negatively charged glutamate residue (V68E) and by a positively charged lysine residue (V68K), With respect to the recombinant wild-type protein the reduction potentials of the V68E and V68K variants are -21+/-2 and + 29+/-2 mV respectively (200 mM NaCl, pH 7, 25 degrees C). The solution structure of the V68E mutant was solved up to a pairwise RMSD of 66 pm for backbone atoms and 138 pm for all heavy atoms. The structure of the variant is very similar to that of recombinant wild type, indicating that the observed changes in reduction potentials are largely due to the effect of the introduced charges. It is proposed that the valence distribution within the oxidized iron-sulfur cluster is affected only slightly by the change in charge at position 68, but consistently with a simple electrostatic model.

1995 - CYCLIC VOLTAMMETRY AND H-1-NMR OF RHODOPSEUDOMONAS-PALUSTRIS CYTOCHROME C(2) - PH-DEPENDENT CONFORMATIONAL STATES [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ferretti, S; Sola, Marco; Soliani, E.
abstract

The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c(2) from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E(o) values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted H-1-NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pK(a) values of 2.5, 5.5, 6.6 and 9.0. The lowest pK(a) most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pK(a) is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pK(a) of 6.6 modulates the E(o) values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s(-1) at pH 9.2.

1995 - CYCLIC VOLTAMMETRY AND H-1-NMR OF RHODOPSEUDOMONAS-PALUSTRIS CYTOCHROME C(2) - PROBING SURFACE-CHARGES THROUGH ANION-BINDING STUDIES [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Dallari, D; Ferretti, S; Sola, Marco
abstract

The effects of increasing concentrations of Cl-, C10(4)(-), and HCO3- on the redox potential of Rhodopseuclonzorzns palustris cytochrome c(2) indicate that the two polyatomic anions bind specifically to the protein at one site, while chloride simply exerts an ionic atmosphere effect. The change in E(o) upon specific anion binding allows us to probe for the influence of surface charges on the redox potential of cytochromes c. The decrease in redox potential at null ionic strength (Delta E(1=0)(o)) due to anion neutralization of one positive surface charge was found to be 23 mV with perchlorate and 33 mV with bicarbonate. These values compare reasonably well with previous theoretical predictions and estimates of the effect of charge alteration on the E(o) values in cytochromes c chemically modified or mutated at surface lysines. These Delta E(o) values, determined on the unmodified protein, are unprecedented for c-type cytochromes. The anion-induced chemical shift changes of the hyperfine-shifted heme H-1-NMR resonances of the oxidized protein yield lower limit values of 53 M(-1) and 18 M(-1) for the affinity constant for specific HCO3- and ClO4- binding, respectively.

1995 - MAGNETIC-RESONANCE OF FE-S CLUSTERS - ISOLATION AND CHARACTERIZATION OF A 7FE FERREDOXIN FROM RHODOPSEUDOMONAS-PALUSTRIS [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; Ferretti, S; Luchinat, C; Sola, Marco
abstract

A novel iron-sulfur protein from the photosynthetic purple bacterium Rhodopseudomonas palustris was purified to homogeneity and identified as a ferredoxin on the basis of its physicochemical properties. Based on the uv/vis spectrum, iron quantitation, cyclic voltammetry, EPR, and H-1 NMR data, the ferredoxin is found to contain two iron-sulfur clusters, one [3Fe-4S] and one [4Fe-4S], which places this protein in the class of 7Fe ferredoxins. The voltammetric peak potentials of the two clusters are -0.260 and -0.560 V at pH 8.0. The molecular mass around 19 kDa makes this protein the heaviest known in this class. This paper further demonstrates the diagnostic power of magnetic resonance spectroscopies in recognition of the two types of clusters in iron-sulfur proteins.

1995 - MUTATION OF THE METAL-BRIDGING PROTON-DONOR HIS63 RESIDUE IN HUMAN CU, ZN SUPEROXIDE-DISMUTASE - BIOCHEMICAL AND BIOPHYSICAL ANALYSIS OF THE HIS63-]CYS MUTANT [Articolo su rivista]
Banci, L; Bertini, I; Borsari, Marco; Viezzoli, Ms; Hallewell, Ra
abstract

The bridging His63 residue in human Cu, Zn superoxide dismutase, which binds both metals, has been replaced by a Cys residue. The mutant protein has been purified from Escherichia coli and appears to be a normal dimer. Spectroscopic techniques (electronic spectroscopies, EPR, nuclear magnetic relaxation dispersion) show that Cys63 binds the zinc ion, but not the copper ion, and that the latter is probably five co-ordinated with three histidine ligands and two water molecules. The reduction potential of the copper ion in the Cu2+/Cu+ pair decreases from 0.41 V to 0.27 V at neutral pH but still remains intermediate between those of the O-2/O-2(-) and O-2(-)/H2O2 pairs so that copper can both oxidize and reduce the O-2(-), substrate, a requirement for dismutase activity. The enzyme binds the substrate-analogue azide (N-3(-)), which displaces one water molecule, with near normal affinity, whereas the enzyme activity with the O-2(-) substrate is reduced to less than 1% of wild-type levels at pH 7.8. The properties of the mutant enzyme are discussed in relation to the superoxide-copper electron transfer process and to the catalytic mechanism.

1995 - Synthesis, Spectroscopic, Magnetic, Conductometric and Electrochemical Investigation of Nickel (II)-1-phenyl-4,6-dimethylpyrimidine-2-thione Complexes [Articolo su rivista]
Battistuzzi, Gianantonio; Borsari, Marco; D., Dallari; Battistuzzi, Raffaele
abstract

Tris-, bis- and mono-ligand complexes of Ni-II with 1-phenyl-4,6-dimethylpyrimidine-2-thione (L) having the general formulae NiL(3)X(2) . 2H(2)O (X=ClO4-, BF4-), NiL(2)X(2) (X = Cl-, Br-, SCN- or NO3-), NiL(2)X(2) . EtOAc (X = Br- or I-), NiL(2)X(2) . H2O . EtOH (X = I- or NO3-) and NiLCl(2) . 3H(2)O, were synthesized and their structures deduced from i.r. and electronic spectra, and magnetic properties. The combined evidence is consistent with an octahedral coordination for the Ni-II ion in all the complexes, with the ligand acting as a bidentate N,S-chelating agent. Spectral evidence, conductivity data and electrochemical results in DMF solution show that the complexes undergo solvolysis readily. Polarographic and c.v. data for the [NiL(3)](ClO4)(2) . 2H(2)O complex and for the [Ni(DMF)(6)](ClO4)(2)-L systems, at increasing ligand concentrations, have shown that in DMF solution the [Ni(DMF)(6)](2+) cation prevails and that the thiopyrimidine-containing species, [NiL(DMF)(5)](2+) (L = N-monodentate ligand) (beta = 2.42 x 10(6)), can be formed only in the presence of a large excess of free ligand.

1995 - THE ROLE OF A CONSERVED TYROSINE RESIDUE IN HIGH-POTENTIAL IRON-SULFUR PROTEINS [Articolo su rivista]
Iwagami, Sg; Creagh, Al; Haynes, Ca; Borsari, Marco; Felli, Ic; Piccioli, M; Eltis, Ld
abstract

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/ Val-58 peptide bond, thereby perturbing Gly-46. The Delta Delta G(D)(app) of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

1994 - ELECTROCHEMICAL REDUCTION OF BENZAMIDE AND THEIR O-HALO-DERIVATIVES AND P-HALO-DERIVATIVES IN NONAQUEOUS SOLVENTS [Articolo su rivista]
Benedetti, Luca; Borsari, Marco; Dallari, D; Fontanesi, Claudio; Grandi, G; Gavioli, Giovanna
abstract

The analysis of the polarographic, voltammetric and coulometric results shows that the overall reduction mechanism of benzamide occurs with the cleavage of the C-N bond. However, the first step of the electrochemical process leads to the formation of a radical anion which undergoes protonation by another benzamide molecule in DMF and DMSO or by the solvent in CH3CN and C2H5OH. Theoretical calculations have been performed to clarify the mechanism. Halobenzamide reduction involves the C-halogen bond breaking as the first reduction step to give benzamide.

1994 - ELECTROCHEMISTRY OF 4,6-DIMETHYL-2-THIOPYRIMIDINE AND 4,6-DIMETHYL-1-PHENYL-2-THIOPYRIMIDINE IN DIMETHYLFORMAMIDE [Articolo su rivista]
Battistuzzi, R; Borsari, Marco; Dallari, D; Gavioli, G; Tavagnacco, C; Costa, G.
abstract

The electrochemical behaviour of 4,6-dimethyl-2-thiopyrimidine (LH) and 4,6-dimethyl-1-phenyl-2-thiopyrimidine (LPHI) and their protonated forms was investigated in dimethylformamide on Hg electrodes. Adsorption processes affect the oxidation mechanism of both the compounds and the formation of Hg(I) complexes was observed. The electrochemical reduction of LH provides an example of the so-called ''father-son reaction'' but this kind of mechanism cannot occur with LPHI. Only in the species LH is a proton present which can be abstracted by the product of the first electron transfer LH.- while LPHI can be protonated by the solvent. However, in the presence of strong acids, for both molecules LH and LPHI the formation of the dimer from the radical anion obtained in the first electron transfer is kinetically preferred to the father-son reaction.

1994 - INFLUENCE OF SURFACE-CHARGES ON REDOX PROPERTIES IN HIGH-POTENTIAL IRON-SULFUR PROTEINS [Articolo su rivista]
Luchinat, C; Capozzi, F; Borsari, Marco; Battistuzzi, Gianantonio; Sola, Marco
abstract

The pH-dependence of the reduction potential determined through differential pulse voltammetry for the high potential iron sulfur proteins (HiPIP) from R. globiformis, C. vinosum, R. gelatinosus, E. vacuolata (I and II), E. halophila (I and II) is reported. A decrease in reduction potential with pH is invariably observed in the pH range where deprotonation of the imidazolium nitrogen of histidine residue(s) occurs. No pH dependence is observed for the only protein lacking histidines. It appears that surface charges like the His imidazolium groups are capable of influencing the reduction potential despite the known quencing of the electrostatic interactions due to solvent effects.

1994 - Magnetic Resonance fingerprints of FeS clusters: isolation and characterization of a Fe7S8 ferredoxin from Rps. palustris [Abstract in Rivista]
Battistuzzi, Gianantonio; Borsari, Marco; S., Ferretti; C., Luchinat; Sola, Marco
abstract

A previously unknown ferredoxin has been isolated and purifiedfrom anaerobic growth of the photosynthetic bacterium Rps. palustris. Cyclic voltammetric experiment gives clear indication of tworedox steps, one around -120 mV and the other around -550 mVversus NHE. H-1 NMR experiments on the three redox statesprovide a clear-cut identification of a [3Fe-4S] and a [4Fe-4S] clusters

1994 - PALLADIUM(II) COMPLEXES OF N-SULFONYLAMINO ACIDS .2. COORDINATION BEHAVIOR UNDER STRONGLY ACIDIC CONDITIONS [Articolo su rivista]
Battistuzzi, Gianantonio; Gavioli, Giovanna; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco
abstract

The ligation of a series of N-sulfonylamino acids (H(2)L) to Pd2+ was investigated by means of d.c. polarography, H-1 NMR and electronic spectroscopy. The first amino acid is found to bind to the metal under extremely acidic conditions, with an apparent pK(NH) value of about 1, while an additional molecule binds with a pK(NH) ranging from 3.1 to 3.7 for the different amino acids. These nitrogen-deprotonated complexes appear not to involve carboxylate complexes as stable precursors. Proton NMR spectroscopy indicates the presence of geometric isomers for the [PdL(2)](2-) species.

1994 - PALLADIUM(II) COMPLEXES OF N-SULFONYLAMINO ACIDS .3. TERNARY ADDUCTS WITH 2,2'-BIPYRIDINE [Articolo su rivista]
Battistuzzi, Gianantonio; Gozzoli, E; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco
abstract

The equilibria involved in the formation of ternary adducts of Pd2+ with N-sulfonylamino acids (H,L) and 2,2'-bipyridine were investigated through d.c. polarography, H-1 NMR and electronic spectroscopy in aqueous solution starting from strongly acidic conditions. The formation of the ternary species appears to proceed through the initial binding of the heteroaromatic base to Pd2+ followed. at higher pH values. by ligation of one amino acid dianion. The pK(NH) value of the latter. ranging from 3.2 to 4.7 for the different amino acids, is unchanged or slightly higher compared to that observed in the formation of the [PdL(2)](2-) species from the precursor [PdL] in the binary Pd2+-L(2-) systems. A comparison is made with the corresponding ternary complexes of Cu2+, Cd2+ and Zn2+.

1994 - Redox equilibria in metalloproteins and characterization of model complexes [Capitolo/Saggio]
Sola, Marco; Borsari, Marco; J. A., Cowan; Menabue, Ledi; Saladini, Monica; Battistuzzi, Raffaele
abstract

Electrochemical and NMR studies of the copper center in superoxide dismutase are described.

1994 - SILOXANE CLUSTERS OF HIGHER VALENCE TRANSITION-METALS - REDOX PROPERTIES [Articolo su rivista]
G., Gavioli; Borsari, Marco; C., Zucchi; Palyi, Gyula; R., Psaro; R., Ugo; O. I., Shchegolikhina; A. A., Zhdanov
abstract

Cyclic voltammetric behaviour of polymetallic complexes of hexaphenylcyclohexasiloxane-hexaol with Ni, Mn and dodecaphenyl-cyclododecasiloxane-dodecaol with Cu was studied. The complexes react electrochemically as a unit assembly of the complexed metals.

1994 - The electronic structure of Fe4S4 clusters [Abstract in Rivista]
L., Banci; Battistuzzi, Gianantonio; I., Bertini; Borsari, Marco; F., Capozzi; S., Ciurli; S., Ferretti; C., Luchinat; M., Piccioli; R., Pierattelli; Sola, Marco
abstract

We have investigated through NMR an extensive series of HiPIPs as well as some ferredoxins in both oxidation states. We have developed a theoretical model for the spin coupling within the cluster that allowed us to rationalize the hyperfine shifts of thecysteine l&and protons as due to the presence of two antierromagnetically coupled spin pairs. The NMR data can thus be interpreted in terms of each individual cysteine beingcoordinated to either an iron belonging to a pair of ferric ions or to a mixed-valence pair.

1994 - Theoretical study of the electroreduction of halogenated aromatic compounds. Part 3. - O-, m- and p-dibromobenzenes studied by AM1 and PM3 methods [Articolo su rivista]
Andreoli, Roberto; Gavioli, Giovanna; Borsari, Marco; Fontanesi, Claudio
abstract

The electroreductive potential values of the o-, m- and p-dibromobenzenes (DBBs) follow an unusual pattern in that, unlike the structurally related dichlorobenzene (DCB) derivatives, these three isomers exhibit a strong 'ortho effect' and are accompanied by a large difference in the E1/2 values. Any interference with the mechanism of reduction on the part of the chemical environment can be safely ruled out, given the consistency of the E1/2 values obtained in four different solvents. The use of theoretical indices calculated by the PM3 method enables the experimental behaviour of the DBBs to be rationalised on the basis of the electronic structure of the neutral isolated molecule. Remarkably, PM3 indicates the formation of a sigma-type radical anion in the case of the reduction of the bromobenzene derivatives.

1993 - Preparation spectroscopic, magnetic, conductometric and polarographic characterization of cobalt(II)-1-phenyl-4,6-dimethylpyrimidine-2-thione complexes [Articolo su rivista]
Borsari, Marco; Battistuzzi, Raffaele; Battistuzzi, Gianantonio
abstract

The following coordination compounds of cobalt(II) with 1-phenyl-4,6-dimethylpyrimidine-2-thione: CoLX2 . n H2O (X = Cl-, n = 1; X = Br-, n = 2; X = SCN-, n = 0; X = NO3-, n = 4), Co2L3Cl4 . H2O, CoL2X2 (X = Br-, I-, SCN-, NO3-), CoL2X2 . n H2O . p Me2CO (X = I-, ClO4-, p = 2; X = NO-, n = 1, p = 0), CoL3(ClO4)2 . 0.5 Me2CO, Co2L5(NO3)4 and Co2L7X4 (X = Cl-, Br-, I-) have been isolated and characterized by chemical analyses, visible and IR spectra, paramagnetic susceptibility, conductivity and polarographic measurements. Solid phase IR spectra suggest that the heterocyclic ligand is almost always bonded through non-substituted ring nitrogen and exocyclic sulfur atoms. The solid compounds with the exception of the [CoL(SCN)2]2, [CoL2(H2O)2][CoCl4] and [CoL3][CoCl4] . H2O have originally a distorted octahedral geometry. By grinding the solid [CoL2(SCN)2], Co2L7Cl4 and all the bromide complexes the cobalt(II) coordination undergo octahedral --> tetrahedral geometry change. Spectral evidences and polarographic results in DMF solution have shown that for these and [CoL(SCN)2]2, [CoL2(H2O)2][CoCl4] and [CoL3][CoCl4] . H2O complexes, the solvolysis gives rise to equilibria between tetrahedral anionic [CoX4]2- (X = SCN-, Cl-), [COX3(DMF)]- (X = SCN-, Cl-, Br-) and some possible [Co(DMF)6]2+, [CoX(DMF)5]+. [CoXL(DMF)4]+, [CoL(DMF)5]2+ (L = N-monodentate ligand) cationic octahedral species. Polarographic data for perchlorate complexes and for [Co(DMF)6](ClO4)2-L system at increasing ligand concentration have shown that in DMF solution the solvated ions (Co(DMF)6]2+ are the prevailing species and that the thiopyrimidine-containing species [CoL(DMF)5]2+ (beta = 3.78 . 10(5)) and (CoL2(DMF)4]2+ (beta = 9.59 . 10(7)) can be formed only in presence or a large excess of the free ligand.

1993 - THEORETICAL-STUDY OF THE ELECTROREDUCTION OF HALOGENATED AROMATIC-COMPOUNDS .2. BROMINE AND CHLORINE DERIVATIVES IN DIFFERENT ORGANIC-SOLVENTS [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Fontanesi, Claudio
abstract

The electrochemical dehalogenation of a variety of halogenated aromatic compounds is studied by means of semiempirical (AM1) and ab initio (ROHF 3-21G*) calculations. Comparison of the electrochemical half-wave potential values (E1/2) and calculated theoretical indices indicates the formation of a pi radical following the electron uptake. Moreover, the relationship observed between the half-wave potential, in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), and the electron affinity suggests that the reduction potential is determined by the first electron uptake and that kinetic factors play a minor role, their influence being constant or negligible.

1992 - REDOX CHEMISTRY OF SUPEROXIDE-DISMUTASE - CYCLIC VOLTAMMETRY OF WILD-TYPE ENZYMES AND MUTANTS ON FUNCTIONALLY RELEVANT RESIDUES [Articolo su rivista]
Azab, Ha; Banci, L; Borsari, Marco; Luchinat, C; Sola, Marco; Viezzoli, Ms
abstract

The reduction potential of human cuprozinc superoxide dismutase and of several of its functionally relevant mutants have been measured through cyclic voltammetry. The reduction potential of the bovine enzyme has been also measured and compared with literature values. The human enzyme has a slightly higher redox potential than the bovine isoenzyme (E-degrees = 0.36 +/- 0.01 and 0.32 +/- 0.01 V vs NHE, at pH 7.4, respectively). The redox properties of the bovine copper-cobalt derivative are very similar to those of the native protein. The pH dependence of the E-degrees value in the wild-type enzyme and its pH independence in the Asn- 1 24 mutant, which has an empty zinc binding site over the entire pH range, is ascribed to the uptake of a proton by His-63 upon reduction. A pK(a) value of 10.8 for this group is obtained from H-1 NMR titrations. It is proposed that also in the zinc-deprived derivative the copper-His-63 bond is broken upon reduction. Sizably negative reduction potentials were estimated for CN- and N3--inhibited enzymes. The values are below the reduction potential of dioxygen to superoxide.

1992 - VOLTAMMETRIC BEHAVIOR OF BOVINE ERYTHROCYTE SUPEROXIDE-DISMUTASE [Articolo su rivista]
Borsari, Marco; Azab, Ha
abstract

VOLTAMMETRIC BEHAVIOR OF BOVINE ERYTHROCYTE SUPEROXIDE-DISMUTASE WAS INVESTIGATED IN AQUEOUS SOLUTION

1992 - Voltammetric behaviour of bovine erythrocyte superoxide dismutase [Articolo su rivista]
Borsari, M.; Azab, H. A.
abstract

1991 - COPPER(II) COMPLEXES WITH N-SULFONYL AMINO-ACIDS - STRUCTURE-STABILITY RELATIONSHIPS IN BINARY SPECIES AND TERNARY COMPLEXES WITH 2,2'-BIPYRIDINE [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco
abstract

The stability of a series of binary and ternary 2,2'-bipyridine(bipy) complexes of Cu2+ with amino acids N-substituted by an arene sulphonic group has been analysed in 0.1 mol dm-3 NaClO4 or NaNO3 at 25-degrees-C. The stability constants determined by d.c. polarography are sensitive to the nature of the amino acid and of the aromatic moiety of the protecting group. The known lowering effect of bipy on the pK for metal-promoted sulphonamide nitrogen deprotonation in the ternary complexes is found to be quantitatively independent of the above structural features. The X-ray structure of the ternary species [Cu(bipy)(Bs-GlyNO)(H2O)] [Bs-GlyNO = N-benzenesulphonylglycinate(2-)] is also presented. The crystals are monoclinic, space group P2(1)/c, with a = 8.494(5), = 19.950(4), c = 11.295(7) angstrom, beta = 110.79(4)-degrees, Z = 4, and R = 0.036. The structure consists of [Cu(bipy)(Bs-GlyNO)(H2O)] units in which the Cu atom exhibits a tetrahedrally distorted square-pyramidal N3O2 coordination.

1991 - Cd2+ and Zn2+ interactions with amino acids N-substituted by a sulfonic group.Effect of the additional ligand 2,2'-bipyridine on the metal-induced amine deprotonation [Articolo su rivista]
Gb, Gavioli; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco
abstract

The interaction of N-(phenylsulfonyl)glycine and N-(tolylsulfonyl)glycine (L) with Cd2+ and Zn2+ was investigated through dc polarography, pH-metric titrations, and H-1 NMR spectroscopy. 2,2'-Bipyridine as additional ligand lowers the pK(a) of Cd2+-promoted deprotonation of the sulfonamide nitrogen (from 8 in the binary system to 7.6 in the presence of the heteroaromatic base), as previously observed for the Cu2+ ion, and, most of all, enables the Zn2+ ion to substitute for the sulfonamide nitrogen bound hydrogen of these ligands. It is known that Zn2+ is ineffective in such a substitution in binary Zn2+-L systems.

1991 - Electrochemical behaviour of oxaziridines at the mercury electrode in organic solvent [Articolo su rivista]
Torre, Giovanni; Moretti, I; Forni, Arrigo; Grandi, G; Fontanesi, Claudio; Borsari, Marco; Gavioli, Gb
abstract

The electrochemical behaviour of 2-methylsulphonyl-3-phenyloxaziridine (1) and 2-t-butyl-3-phenyloxaziridine (2) has been investigated by polarograhic techniques in aprotic solvent. The electroreductive center appears to be the oxaziridine ring but the rearrangement following the electron uptake yields benzaldehyde and imine for the derivatives (1) and (2) respectively. This peculiar behaviour has been discussed on the basis of calculated theoretical indices (CNDO/2 method), like energy values and shapes of frontier molecular orbitals, which suggest the N-O bond breaking as the first step of the overall reduction process.

1991 - POLAROGRAPHIC STUDIES OF CHROMATIUM-VINOSUM HIGH-POTENTIAL IRON PROTEIN - EVIDENCE FOR SURFACE-CHEMISTRY AT THE HG-ELECTRODE [Articolo su rivista]
Borsari, Marco; Sola, Marco; Cowan, Ja
abstract

POLAROGRAPHIC STUDIES OF CHROMATIUM-VINOSUM HIGH-POTENTIAL IRON PROTEIN WERE PERFORMED. EVIDENCE FOR SURFACE-CHEMISTRY AT THE HG-ELECTRODE WAS OBSERVED

1990 - Anodic behaviour of the mercury electrode in aqueous solution of 1,10-phenanthroline. The role of adsorption and nucleation processes [Articolo su rivista]
Andreoli, Roberto; Gavioli, Gb; Benedetti, L; Borsari, Marco; Fontanesi, Claudio
abstract

The anodic dissolution of the mercury electrode in 1,10-phenanthroline (phen) aqueous solution has been investigated by polarographic and voltammetric methods. The adsorption processes affecting the entire mechanism of oxidation and the complex formation have been observed and analysed introducing the random adsorption model (Frumkin isotherm) and the nucleation-growth collision model. The results suggest the occurrence of different surface structures of phenanthroline itself (the ligand) as well as of the product of the oxidative process, [Hg(phen)2]2+, upon varying the ligand concentration in the bulk. © 1990.

1990 - COPPER(II) COMPLEXES WITH 1-PHENYL-4,6-DIMETHYL-PYRYMIDINE-2-THIONE - CHEMICAL, SPECTROSCOPIC, MAGNETIC, CONDUCTIVITY AND POLAROGRAPHIC STUDIES [Articolo su rivista]
Battistuzzi, Raffaele; Borsari, Marco
abstract

COPPER(II) COMPLEXES WITH 1-PHENYL-4,6-DIMETHYL-PYRYMIDINE-2-THIONE WERE STUDIED THROUGH SPECTROSCOPIC, MAGNETIC, CONDUCTIVITY AND POLAROGRAPHIC METHODS

1990 - KINETICS OF COMPACT LAYER FORMATION AND GROWTH OF 1,10-PHENANTHROLINE AT THE ELECTRODE SURFACE [Articolo su rivista]
Benedetti, Luca; Borsari, Marco; Fontanesi, Claudio; Gavioli, Giovanna
abstract

KINETICS OF COMPACT LAYER FORMATION AND GROWTH OF 1,10-PHENANTHROLINE AT THE ELECTRODE SURFACE WERE INVESTIGATED

1990 - Phase transition in the adsorption process of N-Dansylglycinate anion on Mercury [Articolo su rivista]
Benedetti, Luca; Borsari, Marco; Gavioli, Giovanna; Fontanesi, Claudio
abstract

PHASE-TRANSITION IN THE ADSORPTION PROCESS OF N-DANSYLGLYCINATE ANION ON MERCURY WAS STUDIED

1990 - SULFONAMIDE NITROGEN-CONTAINING N-PROTECTED AMINO-ACIDS INTERACTING WITH PALLADIUM(II) - POLAROGRAPHIC AND PH-METRIC INVESTIGATION IN AQUEOUS-SOLUTION [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Pellacani, Gian Carlo; Sola, Marco
abstract

THE BEHAVIOUR OF SULFONAMIDE NITROGEN-CONTAINING N-PROTECTED AMINO-ACIDS INTERACTING WITH PALLADIUM(II) WAS INVESTIGATED THROUGH POLAROGRAPHIC AND PH-METRIC METHODS IN AQUEOUS-SOLUTION

1990 - TERNARY COPPER(II) COMPLEXES WITH 2,2'-BIPYRIDINE AND N-TOSYL-SUBSTITUTED AMINO-ACIDS .1. POLAROGRAPHIC AND PH-METRIC STUDY [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco; Battaglia, Lp; Corradi, Anna; Pelosi, G.
abstract

TERNARY COPPER(II) COMPLEXES WITH 2,2'-BIPYRIDINE AND N-TOSYL-SUBSTITUTED AMINO-ACIDS WERE INVESTIGATED THROUGH POLAROGRAPHIC AND PH-METRIC METHODS

1990 - TERNARY COPPER(II) COMPLEXES WITH 2,2'-BIPYRIDINE AND N-TOSYL-SUBSTITUTED AMINO-ACIDS .2. CRYSTAL AND MOLECULAR-STRUCTURE OF AQUA(2,2'-BIPYRIDINE)BIS(N-TOSYL-DL-ASPARAGINATO-O)COPPER(II) DIHYDRATE AND (2,2'-BIPYRIDINE)(N-TOSYL-DL-ASPARAGINATO-NO)COPPER(II) [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Menabue, Ledi; Saladini, Monica; Sola, Marco; Battaglia, Lp; Corradi, Anna; Pelosi, G.
abstract

CRYSTAL AND MOLECULAR-STRUCTURE OF AQUA(2,2'-BIPYRIDINE)BIS(N-TOSYL-DL-ASPARAGINATO-O)COPPER(II) DIHYDRATE AND (2,2'-BIPYRIDINE)(N-TOSYL-DL-ASPARAGINATO-NO)COPPER(II) WERE DETERMINED

1989 - Electrochemical behaviour of N-Tosylglycine and N-Dansylglycine in several solvents. The role of the RSO2- groups on the physico-chemical properties of Glycine [Articolo su rivista]
Fontanesi, Claudio; Borsari, Marco; Andreoli, A.; Benedetti, L.; Grandi, G.; Gavioli, G.
abstract

The comparison of the electrochemical behaviour of N-tosylglycinoa nd N-dansylglycinew withthat of the corresponding amides and the benzenesulphonamidine in protic and aprotic media has shown thatthe RSO2- protected aminoacids, in their neutral form, can be considered as benzenesulphonamidederivatives and their E1/2 values fit the structural correlations among experimental and theoreticalparameters already discussed in previous papers. Using these correlations, the role of the RSO2- andcarboxylate groups on the amidic acidity of the free and complexed aminoacid has been determined also onthe basis of calculated differences in total energy of dissociated and undissociated acidic species. Allthese results confirm the main role played by the carboxylate group on the formation of N-O bidentatecomplexes.

1989 - The effect of lateral chains on the electrochemical and physico-chemical behaviour of Tosyl-N-protected aminoacids [Articolo su rivista]
Benedetti, Luca; Borsari, Marco; Fontanesi, Claudio; Grandi, Giulia; Andreoli, Roberto
abstract

The electrochemical behaviour in protic and aprotic solvents of some tosylaminoacids bearing a lateral electroinactive chain confirms that the reduction mechanism conforms to that one already proposed for tosyl- and dansylglycine and benzenesulphonamide derivatives. The E value reveals to be a good structural index and relationships between empirical and calculated structural parameters show that the presence of the carboxylate group in tosylaminoacids causes an increase of NH bond acidity with respect to benzenesulphonamide derivatives. The pK values of the amide group are influenced by the kind of the lateral chains and by the position of carboxylic group in the molecular frame.

1988 - Effectiveness of the cadmium(II) ion in promoting nitrogen deprotonation. 113 Cd NMR, polarographic, and pH-metric investigations on the Cadmium(II)-N-tosyl-glycinate and Cadmium(II)-N-dansylglycinate systems in aqueous and methanolic systems [Articolo su rivista]
Gavioli, Giovanna; Borsari, Marco; Pellacani, Gian Carlo; Menabue, Ledi; Sola, Marco
abstract

13Cd NMR, polarographic, and pH-metric investigations reveal the ability of the Cd2' ion in substituting for the nitrogen-boundhydrogen of the amino acids N-protected by a sulfonic group. In aqueous and methanolic solution, N-tosylglycine and Ndansylglycine interacting with the Cd2+ ion show a pH-dependent binding mode, at increasing pH changing from simple carboxylate to N,O-bidentate ligands. The overall equilibria involving the prevailing complexes are similar to those previously found with the Cu2+ ion, but the stability of the cadmium complexes is lower by factors ranging from IO to lo4. Two binary complexes of formula [CdL,(H,O),] (L = N-tosylglycinate, N-dansylglycinate) were separated in the solid state. X-ray powder spectra and IR data for the complex of N-tosylglycine show that it is isomorphous and isostructural with the analogous complexes with Zn2', Co2+, and Ni2+, in which the ligand is monodentate through the carboxylate group.

1988 - Electrochemical behaviour of Chlorobenzene sulphonamide derivatives at the Mercury electrode in non aqueous solvents [Articolo su rivista]
Borsari, Marco; Fontanesi, Claudio; Andreoli, R.; Benedetti, L.; Gavioli, G.
abstract

The analysis of polarographic and coulometric results shows that the overall reductionmechanism of chlorobenzenesulphonamide derivatives occurs with the cleavage of the C-Cl bond followed bythe reduction of the benzenesulphonamide involving the S-N bond breaking. However, theg, ,2 values fit thestructural relation E, ,2-ELUMo for the series of benzenesulphonamides and not that of the chlorobenzenederivatives and the shape of the LUMO of chlorobenzenesulphonamides is similar to that of thebenzenesulphonamide, so that it is sensible to suggest that the first step of the reduction mechanism is thesame for all the benzenesulphonamide derivatives and involves the C-S-N moiety of the molecules.