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Antonio RANIERI

Professore Associato
Dipartimento di Scienze della Vita sede ex Chimica V.Campi 103

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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.

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 - 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 - 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 - 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 - 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 - 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 - 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 - 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.

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 - 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.

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 - PRELIMINARY OBSERVATIONS ON SCLERAL OSSICLES IN PERFORMING FUNCTIONALIZED 3D VASCULARIZED SCAFFOLDS FOR "CRITICAL_SIZE" BONE DEFECT HEALING [Abstract in Rivista]
Checchi, Marta; Smargiassi, Alberto; Ferretti, Marzia; Sena, Paola; Benincasa, Marta; Cavani, Francesco; Sola, Marco; Ranieri, Antonio; Stefania, Mitola; Palumbo, Carla
abstract

PRELIMINARY OBSERVATIONS ON SCLERAL OSSICLES IN PERFORMING FUNCTIONALIZED 3D VASCULARIZED SCAFFOLDS FOR "CRITICAL_SIZE" BONE DEFECT HEALING

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 - 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 - 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.

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 - Enhancing Biocatalysis: The Case of Cytochrome c Unfolded Immobilized on Kaolinite [Articolo su rivista]
Castellini, Elena; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Bernini, Fabrizio; Ranieri, Antonio
abstract

Urea-unfolded wild-type cytochrome c and its variants immobilized on kaolinite show peroxidase activity that is significantly higher than that of the folded wild-type protein. The accessibility of the substrate to the metal center and the influence of strategic amino acidic residues on the surface of the protein are discussed. This approach sheds light on the factors affecting the catalytic activity of a new versatile biocatalytic interface.

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 - 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.

2012 - The Met80Ala point mutation enhances the peroxidase activity of immobilized cytochrome c [Articolo su rivista]
Ranieri, Antonio; Bernini, Fabrizio; Bortolotti, Carlo Augusto; Castellini, Elena
abstract

The effects of replacement of the axial methionine 80 hemeligand with a non-coordinating alanine on the peroxidaseactivity of kaolinite-immobilized cytochrome c wereinvestigated at different pH values. The catalytic activity10 of the adsorbed mutant was found remarkably higher thanthat of wild-type cytochrome c. The pH dependence ofVmax and KM values is discussed in term of accessibility ofthe substrates to the metal center and surface charge ofkaolinite. Our approach, based on the combined use of15 adsorption on kaolinite and protein engineering, endowsthis bioinorganic interface with remarkable catalyticproperties.

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.

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 - pH-dependent peroxidase activity of yeast cytochrome c and its triple mutant adsorbed on kaolinite [Articolo su rivista]
Ranieri, Antonio; Bernini, F.; Bortolotti, Carlo Augusto; Bonifacio, A.; Sergo, V.; Castellini, Elena
abstract

The peroxidase activity of wild type yeast cytochrome c and its triple mutant K72AK73AK79A adsorbed onto kaolinite was investigated as a function of pH and temperature. Both adsorbedproteins displayed an appreciable catalytic activity, which remained constant from pH 7 to 10, decreased below pH 7 and showed a remarkable increase at pH values lower than 4. In the whole pH range investigated the catalytic activity of the adsorbed wild type cytochrome c was higher than that of the mutant. Both diffuse-reflectance UV-Vis and Resonance Raman spectroscopies applied on solid samples were used to probe the structural features responsible for the catalytic activity of the immobilized proteins. At neutral and alkaline pH values a six-coordinate low-spin form of cytochrome c was observed, while at pH < 7 the formation of a high-spin species occurred whose population increased at decreasing pH. The orientation and exposure of the haem to the substrate - strictly dependent on adsorption - was found to affect the peroxidase activity.

2011 - The Reorganization Energy in Cytochrome c is Controlled by the Accessibility of the Heme to the Solvent [Articolo su rivista]
Bortolotti, Carlo Augusto; Siwko, Magdalena Elzbieta; Castellini, Elena; Ranieri, Antonio; Sola, Marco; S., Corni
abstract

Elucidation of the molecular determinants of the reorganization energy λ is central to the understanding of fundamental biological processes based on energy transduction pathways. Here, we use a combined experimental/theoretical approach to electrochemically determine the reorganization energy for a number of cytochrome c variants and compute structure-related properties relevant to the kinetics of the electron transfer process through molecular dynamics simulations. We find that the exposure of the heme group to solvent controls the reorganization energy of the investigated proteins. Therefore, fine-tuning of the kinetics of the electron transfer process can be achieved through modulation of the accessibility of the iron to the surrounding water. Our findings lead the way for a new strategy for the design of protein-based bioelectronic materials, requiring fast and efficient electron transfer.

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 - 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 - Thermodynamic Aspects of the Adsorption of Cytochrome c and Its Mutants on Kaolinite [Articolo su rivista]
Castellini, Elena; Ranieri, Antonio; Domenico A., Simari; Di Rocco, Giulia
abstract

The adsorption of native, wild-type and engineered cytochrome c on sodium-exchanged kaolinite was investigated by spectroscopic means. The variants of yeast cytochrome c were obtained replacing surface lysines in position 72, 73 and 79 with alanine residues. All proteins are strongly adsorbed onto kaolinite. In particular, the presence of the lysine residue in position 73 remarkably favors adsorption. A detailed characterization of the thermodynamic aspects of the adsorption process has been performed. Most notably, adsorbed cytochrome c maintains its moderate peroxidase activity against guaiacol. This investigation is prodromal to the exploitation of the catalytic activity of engineered cytochrome c immobilized on a polydisperse system.

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 - 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. --------------------------------------------------------------------------------

2007 - Protonation of a Histidine Copper Ligand in Fern Plastocyanin [Articolo su rivista]
R., Hulsker; A., Mery; E. A., Thomassen; Ranieri, Antonio; Sola, Marco; Verbeet, M. P. H.; T., Kohzuma; M., Ubbink
abstract

Plastocyanin is a small blue copper protein that shuttles electrons as part of the photosynthetic redox chain. Its redox behavior is changed at low pH as a result of protonation of the solvent-exposed copper-coordinating histidine. Protonation and subsequent redox inactivation could have a role in the down regulation of photosynthesis. As opposed to plastocyanin from other sources, in fern plastocyanin His90 protonation at low pH has been reported not to occur. Two possible reasons for that have been proposed: - stacking between Phe12 and His90 and lack of a hydrogen bond with the backbone oxygen of Gly36. We have produced this fern plastocyanin recombinantly and examined the properties of wild-type protein and mutants Phe12Leu, Gly36Pro, and the double mutant with NMR spectroscopy, X-ray crystallography, and cyclic voltammetry. The results demonstrate that, contrary to earlier reports, protonation of His90 in the wild-type protein does occur in solution with a pKa of 4.4 (±0.1). Neither the single mutants nor the double mutant exhibit a change in protonation behavior, indicating that the suggested interactions have no influence. The crystal structure at low pH of the Gly36Pro variant does not show His90 protonation, similar to what was found for the wild-type protein. The structure suggests that movement of the imidazole ring is hindered by crystal contacts. This study illustrates a significant difference between results obtained in solution by NMR and by crystallography.

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.

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.

2006 - Towards combined electrochemistry and surface-enhanced resonance Raman of heme proteins: Improvement of diffusion electrochemistry of cytochrome c at silver electrodes chemically modified with 4-mercaptopyridine [Articolo su rivista]
D., Millo; Ranieri, Antonio; W., Koot; C., Gooijer; G., van der Zwan
abstract

To date, a successful combination of surface-enhanced resonance Raman spectroscopy (SERRS) and electrochemistry to study heme proteins is inhibited by the problems raised by the prerequisite to use silver as electrode metal. This paper indicates an approach to overcome these problems. It describes a quick and reproducible procedure to prepare silver electrodes chemically modified with 4-mercaptopyridine suitable to perform diffusion electrochemistry of cytochrome c (cyt c). The method involves the employment of a mechanical and a chemical treatment and avoids the use of alumina slurries and any electrochemical pretreatment. Cyclic voltammetry (CV) was used to test the electrochemical response of cyt c, and the CV signals were found identical with those obtained on gold electrodes under the same experimental conditions. Compared to previous literature, a significant improvement of the CV signal of cyt c at silver electrodes was achieved. Preliminary results show that this treatment can be also successfully employed for the preparation of SERRS-active electrodes.

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 - 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 - 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 - Protein stability and mutations in the axial methionine loop of a minimal cytochrome c [Articolo su rivista]
I., Bartalesi; I., Bertini; DI ROCCO, Giulia; Ranieri, Antonio; A., Rosato; M., Vanarotti; P. R., Vasos; M. S., Viezzoli
abstract

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.

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 - 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 - 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 &gt; 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 - 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 - 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 - 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 - 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 - 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.