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Gianantonio BATTISTUZZI

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

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Pubblicazioni

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

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

2024 - Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants [Articolo su rivista]
Gabler, Thomas; Dali, Andrea; Bellei, Marzia; Sebastiani, Federico; Becucci, Maurizio; Battistuzzi, Gianantonio; Georg Furtmüller, Paul; Smulevich, Giulietta; Hofbauer, Stefan
abstract

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria, in 2015 by Dailey and coworkers triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely H182A and E263Q, involving two key active site residues. Interestingly, both variants are active only towards the physiological substrate coproporphyrin III but inactive towards protoporphyrin IX. In addition, E263 is impairing the final oxidation from ferrous coproheme to ferric coproheme. The characteristics of the active site in terms of the residues involved and the substrate binding properties are discussed by structural and functional means, providing a further contribution to the deciphering of the enigmatic reaction mechanism.

2023 - Compound I formation and reactivity in dimeric chlorite dismutase – Impact of pH and the dynamics of the catalytic arginine [Articolo su rivista]
Schmidt, Daniel; Falb, Nikolaus; Serra, Ilenia; Bellei, Marzia; Pfanzagl, Vera; Hofbauer, Stefan; Van Doorslaer, Sabine; Battistuzzi, Gianantonio; Furtmüller, Paul; Obinger, Christian
abstract

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

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 - Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida [Articolo su rivista]
Serra, Ilenia; Piccinini, Daniele; Paradisi, Alessandro; Ciano, Luisa; Bellei, Marzia; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Sola, Marco; Walton, Paul H.; DI ROCCO, Giulia
abstract

Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.

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

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

2021 - 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.

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

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

2019 - Redox thermodynamics of B-class dye-decolorizing peroxidases [Articolo su rivista]
Pfanzagl, Vera; Bellei, Marzia; Hofbauer, Stefan; Laurent, Christophe V. F. P.; Furtmüller, Paul G.; Oostenbrink, Chris; Battistuzzi, Gianantonio; Obinger, Christian
abstract

With>5000 annotated genes dye-decolorizing peroxidases (DyPs) represent a heme b peroxidase family of broad functional diversity. Bacterial B-class DyPs are poor peroxidases of unknown physiological function. Hydrogen peroxide efficiently mediates the rapid formation of Compound I in B-class DyPs, which, however, is stable and shows modest reactivity towards organic and inorganic electron donors. To understand these characteristics, we have investigated the redox thermodynamics of the one-electron reduction of the ferric high-spin form of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) and the variants D143A, R232A and D143A/R232A. These distal amino acids are fully conserved in all DyPs and play important roles in Compound I formation and maintenance of the heme cavity architecture and substrate access route(s). The E°′ values of the respective redox couples Fe(III)/Fe(II) varied from −350 mV (wild-type KpDyP) to −299 mV (D143A/R232A) at pH 7.0. Variable-temperature spectroelectrochemical experiments revealed that the reduction reaction of B-class DyPs is enthalpically unfavored but entropically favored with significant differences in enthalpic and entropic contributions to E°′ between the four proteins. Molecular dynamics simulations demonstrated the impact of solvent reorganization on the entropy change during reduction reaction and revealed the dynamics and restriction of substrate access channels. Obtained data are discussed with respect to the poor peroxidase activities of B-class DyPs and compared with heme peroxidases from other (super)families as well as with chlorite dismutases, which do not react with hydrogen peroxide but share a similar fold and heme cavity architecture.

2018 - Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage [Articolo su rivista]
Pfanzagl, Vera; Nys, Kevin; Bellei, Marzia; Michlits, Hanna; Mlynek, Georg; Battistuzzi, Gianantonio; Djinovic-Carugo, Kristina; Van Doorslaer, Sabine; Furtmüller, Paul G.; Hofbauer, Stefan; Obinger, Christian
abstract

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high resolution crystal structures of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp 143 and Arg 232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin (LS) cyanide complex as well as on (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527 and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

2018 - Roles of the distal Asp-Arg pair in Compound I formation of K. pneumoniae dye-decolorizing peroxidase [Abstract in Rivista]
Pfanzagl, Vera; Hofbauer, Stefan; Nys, Kevin; Djinovic-Carugo, Kristina; Battistuzzi, Gianantonio; Van Doorslaer, Sabine; Furtmuller, Paul G.; Obinger, Christian
abstract

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6A˚ ) and the variants D143A (1.3 A˚ ), R232A (1.9 A˚ ), and D143A/R232A (1.1 A˚ ). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin-cation radical (Compound I).

2018 - Secreted Heme Peroxidase from Dictyostelium discoideum: Insights into Catalysis, Structure and Biological Role [Articolo su rivista]
Nicolussi, Andrea; Dan Dunn, Joe; Mlynek, Georg; Bellei, Marzia; Zamocky, Marcel; Battistuzzi, Gianantonio; Djinović-Carugo, Kristina; Furtmüller, Paul G.; Soldati, Thierry; Obinger, Christian
abstract

Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum. This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a posttranslationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and E236. The metalloprotein follows the halogenation cycle, whereby Compound I oxidizes iodide and thiocyanate at high (> 108 M-1 s-1) and bromide at very low rates. It is demonstrated that DdPoxA is upregulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared to evolutionary related mammalian peroxidases in the context of non specific immune defense.

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.

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

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

2017 - Molecular mechanism of enzymatic chlorite detoxification: insights from structural and kinetic studies [Articolo su rivista]
Schaffner, Irene; Mlynek, Georg; Flego, Nicola; Puehringer, Dominic; Libiseller egger, Julian; Coates, Leighton; Hofbauer, Stefan; Bellei, Marzia; Furtmüller, Paul; Battistuzzi, Gianantonio; Smulevich, Giulietta; Djinovic carugo, Kristina; Obinger, Christian
abstract

The heme enzyme chlorite dismutase (Cld) degrades chlorite to chloride and dioxygen. Although the structure and steady-state kinetics of pentameric Clds have been elucidated, many questions remain, such as the mechanism of chlorite cleavage and the pH dependence of the reaction. Here, we present high resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple and the UV-vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite cannot recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during reaction. Homolytic cleavage of chlorite is proposed

2017 - Posttranslational Modification of Heme b in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis [Articolo su rivista]
Nicolussi, Andrea; Auer, Markus; Weissensteiner, Julia; Schuetz, Georg; Katz, Sonja; Maresch, Daniel; Hofbauer, Stefan; Bellei, Marzia; Battistuzzi, Gianantonio; Furtmüller, Paul; Obinger, Christian
abstract

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases including myeloperoxidase (MPO) and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. The present study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A and D109A/E238A which could be recombinantly produced as apo-proteins in E. coli, fully reconstituted to the respective heme b proteins and posttranslationally modified by hydrogen peroxide. This for the first time allows not only to directly compare the catalytic properties of the heme b and PTM forms, but also to study the impact of D109 and E238 on PTM and catalysis including Compound I formation and the two-electron reduction of Compound I by bromide, iodide and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238 - in contrast to exchange of D109 - does not cause significant structural rearrangements and changes of the catalytic properties neither in the heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.

2017 - Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1 [Articolo su rivista]
Paumann Page, Martina; Katz, Romy Sophie; Bellei, Marzia; Schwartz, Irene; Edenhofer, Eva; Sevcnikar, Benjamin; Soudi, Monika; Hofbauer, Stefan; Battistuzzi, Gianantonio; Furtmueller, Paul G.; Obinger, Christian
abstract

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid which mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here we present the first multi-mixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immune-globulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle which is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate Compound I. We demonstrate that chloride cannot act as two-electron donor of Compound I, whereas thiocyanate, iodide and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates and the standard reduction potential of the Fe(III)/Fe(II) couple and we demonstrate that the prosthetic heme group is posttranslationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid which mediates the formation of covalent crosslinks in collagen IV.

2016 - Chemistry and Molecular Dynamics Simulations of Heme b‑HemQ and Coproheme-HemQ [Articolo su rivista]
Hofbauer, Stefan; Dalla Sega, Marco; Scheiblbrandner, Stefan; Jandova, Zuzana; Schaffner, Irene; Mlynek, Georg; Djinović Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Oostenbrink, Chris; Obinger, Christian
abstract

Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading fromcoproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of −205 ± 3 mV for LmHemQ and −207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 104 M−1 s−1). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria.

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 - Vinca il migliore. L’agonismo nel mondo naturale dal nucleo atomico alla scala cosmica [Esposizione]
Brunetti, Rossella; Battistuzzi, Gianantonio; Malavasi, Giancarlo; Bosi, Giovanna; Barbieri, Giovanna
abstract

Il progetto 2016 del ciclo La Curiosità fa lo Scienziato ha proposto una riflessione sui temi della competizione e collaborazione nel mondo naturale, coinvolgendo gli ambiti disciplinari di fisica, chimica e scienze naturali. In tutto il mondo naturale osservabile equilibri e dinamiche sono riconducibili ad entità in competizione, secondo regole che si vanno scoprendo man mano che aumenta la capacità di interrogare la natura. In fisica entrano nell’arena poche forze fondamentali, dalla micro scala atomica fino alla macro scala cosmologica. In chimica diversi composti competono nel determinare l’evoluzione e gli effetti delle reazioni. Nel mondo biologico il rapporto che si stabilisce in un determinato ambiente tra le varie specie animali e vegetali, legate tra loro da relazioni antagonistiche, ma anche mutualistiche, è condizione indispensabile per la sopravvivenza. Gli incontri previsti all’interno del Progetto, dedicati a platee di età diverse, hanno sempre incluso conversazioni a tema arricchite da esperimenti includendo in talune circostanze anche esecuzioni di brani musicali. Le attività hanno complessivamente raggiunto un pubblico di circa 1200 persone. Nel seguito vengono elencate e brevemente descritte le varie attività svolte. Il materiale documentale prodotto durante il Progetto è messo a disposizione a libero accesso sul sito del Dipartimento di Scienze Fisiche, Informatiche e Matematiche dell’Università di Modena e Reggio Emilia al link: http://www.outreach.fim.unimore.it/site/home/divulgazione/la-curiosita-fa-lo-scienziato

2015 - Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425 [Articolo su rivista]
Schaffner, Irene; Hofbauer, Stefan; Krutzler, Michael; Pirker, Katharina F.; Bellei, Marzia; Stadlmayr, Gerhard; Mlynek, Georg; Djinovic Carugo, Kristina; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Daims, Holger; Obinger, Christian
abstract

It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) may 34 contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite “dismutase”, Cld). Beside the water-splitting manganese complex of photosystem II this metalloenzyme is the second known enzyme that catalyzes the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in E. coli and shown to efficiently degrade chlorite with an activity optimum at pH 5 (kcat 1144 ± 23.8 s-1, KM 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 106 M-1 s-1). The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7. Cyanide mediates the formation of a low-spin complex with kon = (1.6 ± 0.1) × 105 M-1 s-1 and koff = 1.4 ± 2.9 s-1 (KD ~ 8.6 μM). Both, thermal and chemical unfolding follows a non-two state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2-producing proteins in (nitrogen-fixing) cyanobacteria.

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 - Multidomain human peroxidasin 1 is a highly glycosylated and stable homotrimeric high-spin ferric peroxidase [Articolo su rivista]
Soudi, Monika; Delporte, Cedric; Paumann Page, Martina; Pirker, Katharina F.; Bellei, Marzia; Edenhofer, Eva; Stadlmayr, Gerhard; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Van Antwerpen, Pierre; Obinger, Christian; Boudjeltia, Karim Zouaoui
abstract

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine crosslinks. The latter confer critical structural reinforcement to collagen IV scaffolds. Here hsPxd01 and various truncated variants lacking non-enzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure and unfolding pathway are reported. The homotrimeric ironprotein contains a covalently-bound ferric high-spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of -233 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KM) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV crosslinking.

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 - Chlorite to chloride and O2 conversion: new lessons from structural and mechanistic investigations of chlorite dismutase [Abstract in Rivista]
C., Obinger; S., Hofbauer; I., Schaffner; K. F., Pirker; G., Mlynek; K., Djinovic Carugo; Battistuzzi, Gianantonio; P. G., Furtmuller
abstract

Oral Presentation

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

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

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

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

2014 - Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry and Reactivity [Articolo su rivista]
Stefan, Hofbauer; Kira, Gysel; Bellei, Marzia; Andreas, Hagmüller; Irene, Schaffner; Georg, Mlynek; Julius, Kostan; Katharina F., Pirker; Holger, Daims; Paul G., Furtmüller; Battistuzzi, Gianantonio; Kristina Djinović, Carugo; Christian, Obinger
abstract

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “Candidatus Nitrospira defluvii” (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV–vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O═O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°′ = −113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.

2014 - Mechanistic studies on a new functional dimeric chlorite dismutase [Abstract in Rivista]
I., Schaffner; N., Flego; G., Mlynek; Bellei, Marzia; S., Hofbauer; Battistuzzi, Gianantonio; K., Djinovic Carugo; G., Smulevich; P. G., Furtmuller; C., Obinger
abstract

Poster presentation

2014 - New highly stable chlorinating bacterial peroxidase with autocatalytically formed covalent heme to protein bonds [Abstract in Rivista]
P. G., Furtmuller; M., Auer; C., Gruber; K., Pirker; D., Krioss; S., Hofbauer; M., Soudi; C., Obinger; M., Zamocky; Bellei, Marzia; Battistuzzi, Gianantonio
abstract

Here, for the first time, an ancestral bacterial heme peroxidase has been cloned and purified. It is shown to catalyse (besides conventional peroxidase activity) bromide and chloride oxidation more efficiently than LPO and MPO. The structure-function relationships of this new peroxidase in relation to its mammalian counterparts and its putative physiological role are discussed.

2014 - The role of covalent heme to protein bonds in the formation and reactivity of redox intermediates of a bacterial peroxidase with high homology to human peroxidases [Abstract in Rivista]
P. G., Furtmuller; M., Auer; A., Nicolussi; G., Schutz; Bellei, Marzia; Battistuzzi, Gianantonio; C., Obinger
abstract

Oral presentation

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

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

2014 - Understanding Chlorite Dismutase from Candidatus Nitrospira defluvii [Abstract in Rivista]
S., Hofbauer; C., Gruber; K. F., Pirker; I., Schaffner; P. G., Furtmuller; C., Obinger; A., Hagmuller; K., Gysel; G., Mlynek; J., Kostan; K., Djinovic Carugo; Bellei, Marzia; Battistuzzi, Gianantonio; H., Daims
abstract

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “Candidatus Nitrospira defluvii” (NdCld) were performed, using UV-vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics as well as X-ray crystallography. The results obteined are described.

2013 - A stable bacterial peroxidase with novel halogenating activity and an autocatalytically linked heme prosthetic group [Articolo su rivista]
Auer, Markus; Gruber, Clemens; Bellei, Marzia; Pirker, Katharina F.; Zamocky, Marcel; Kroiss, Daniela; Teufer, Stefan A.; Hofbauer, Stefan; Soudi, Monika; Battistuzzi, Gianantonio; Furtmüller, Paul G.; Obinger, Christian
abstract

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase (LPO) and myelo-peroxidase (MPO) revealed the presence of new bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to catalyse (besides conventional peroxidase activity) bromide and chloride oxidation more efficiently than LPO and MPO. The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby the high-spin ferric rhombic heme spectrum became similar to LPO, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (-145 ± 10 mV at pH 7) and the conformational and thermal stability of the protein increased. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.

2013 - Attrazioni fatali: Legami invisibili che danno forma all’Universo [Esposizione]
Brunetti, Rossella; Battistuzzi, Gianantonio
abstract

Il Dipartimento FIM ha partecipato al Festival Filosofia 2013 sull’amare con una installazione scientifica interattiva dal titolo: ATTRAZIONI FATALI - Legami invisibili che danno forma all’universo “Amore è tutta questione di chimica. Ma se con il tuo sguardo magnetico vieni respinto… o non sei attratto da nessuno… Prova con un buco nero!” L’installazione, a ingresso libero e ospitata dalla Biblioteca Delfini dalle 15 alle 20 nei giorni 13, 14 e 15 settembre 2013, è nata da una collaborazione tra UniMore, l’Associazione Inco.SCIENZA e la Biblioteca Delfini e ha trattato i temi: gravità, interazioni elettromagnetiche, legami tra particelle. Coordinatori scientifici sono stati Rossella Brunetti (Dip. di Scienze Fisiche, Informatiche e Matematiche) e Gianantonio Battistuzzi (Dip. di Chimica e Scienze della Terra). Attraverso esperimenti, filmati, giochi e poster a tema i visitatori sono stati condotti a gettare uno sguardo su attrazioni e repulsioni nel linguaggio della fisica e della chimica per sperimentare una nuova chiave interpretativa di noi stessi e di ciò che sta fuori di noi. Attrazioni o repulsioni non sono solo relazioni umane: esistono alle radici del mondo osservabile e sono dentro di noi, determinano la nostra percezione della realtà e il nostro essere all’interno del mondo naturale. I fisici le chiamano “forze”. Attrazione fatale della mela verso la terra, ma anche della Terra verso il nostro sole e, ancora oltre, del nostro sole al cuore pulsante della nostra galassia. L’ assenza di questa attrazione fatale, che crea un “sopra” e un “sotto”, un “alto” e un “basso” nella nostra vita quotidiana, ci consente di allontanarci da noi stessi e vedere la dimensione umana da un orizzonte più vasto sul quale la fragilità e l’unicità dell’essere umano si contemplano con orgoglio e con sgomento assieme. Poi altre attrazioni e repulsioni fatali plasmano la materia, creano o distruggono legami chimici, forniscono vita alle nostre cellule. Nel mondo degli atomi che ci compongono, ora meno segreto, c’è l’”amore” , cioè l’attrazione, ma anche l’”odio”, il suo opposto, la repulsione. Come nelle umane vicende, anche nella fisica amore e odio, attrazione e repulsione, dettano regole e stabiliscono rapporti, influenzano comportamenti e prospettano possibili scenari. Le regole del gioco questa volta sono chiare, prevedibili, descritte dal linguaggio universale della matematica. Gettate uno sguardo assieme a noi sull’amore e l’odio nel mondo della fisica: il vostro! L’installazione ha presentato poster illustrativi, caroselli di diapositive a tema, piccoli filmati significativi, e due “tavoli sperimentali” di semplici esperienze da realizzare con il pubblico sotto la guida di atelieristi a tema: “gravità” e “interazioni elettromagnetiche”, secondo il seguente schema.

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

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

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

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

2013 - 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 - Eukaryotic Extracellular Catalase-Peroxidase from Magnaporthe grisea – Biophysical/Chemical Characterization of the First Representative from a Novel Phytopathogenic KatG Group [Articolo su rivista]
M., Zámocký; E., Droghetti; Bellei, Marzia; B., Gasselhuber; M., Pabst; P. G., Furtmüller; Battistuzzi, Gianantonio; G., Smulevich; C., Obinger
abstract

All phytopathogenic fungi have two catalaseeperoxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologouslywith almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UVeVis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°' of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanidewas relatively slow at pH 7.0 and 25 °C and with a Kd value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure-function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host-pathogen interaction.

2012 - Redox thermodynamics of high-spin and low-spin forms of chlorite dismutases of diverse subunit and oligomeric structure [Articolo su rivista]
S., Hofbauer; Bellei, Marzia; A., Sündermann; K. F., Pirker; A., Hagmüller; G., Mlynek; J., Kostan; H., Daims; P. G., Furtmüller; K., Djinović Carugo; C., Oostenbrink; Battistuzzi, Gianantonio; C., Obinger
abstract

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus “Nitrospira defluvii” (NdCld) were determined through spectroelectrochemical experiments. The above proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 kDa versus 26 kDa) and oligomeric (dimeric versus pentameric) structure but exhibit similar chlorite degradation activity. The E°’ values for free and cyanide-bound proteins were determined to be -119 mV and -397 mV for NwCld as well as -113 mV and -404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase of entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.

2012 - Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria ofCytochrome c [Articolo su rivista]
Battistuzzi, Gianantonio; Bortolotti, Carlo Augusto; Bellei, Marzia; DI ROCCO, Giulia; J., Salewski; P., Hildebrandt; Sola, Marco
abstract

The low-pH conformational equilibria of ferricyeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, andM80A/Y67A variants were studied from pH 7 to 2 at low ionicstrength through electronic absorption, magnetic circulardichroism, and resonance Raman spectroscopies. For wild-typeycc, the protein structure, axial heme ligands, and spin state ofthe iron atom convert from the native folded His/Met low-spin(LS) form to a molten globule His/H2O high-spin (HS) formand a totally unfolded bis-aquo HS state, in a single cooperativetransition with an apparent pKa of ∼3.0. An analogouscooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pKa of ∼4.2, and by an equilibrium between a His/OH−-ligated LS and a His/H2O-ligated HS conformer, with a pKa of∼5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increasedstability of the molten globule state that is formed at higher pH values than the other species. These data show that removal ofthe axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pHconformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule andrenders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the threedimensionalstructure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues71−85 and 40−57.

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

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

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

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

2011 - Bifunctional catalase-peroxidase is secreted by the rice blast fungus Magnaporthe grisea during oxidative burst [Abstract in Rivista]
M., Zamocky; B., Gasselhuber; P. G., Furtmueller; Bellei, Marzia; Battistuzzi, Gianantonio; C., Obinger
abstract

Recently, it has been demonstrated that secreted bifunctional catalase-peroxidase is essential for the survival of the rice blast fungus Magnaporthe grisea during oxidative burst induced by theinfected plant (Tanabe et al., Mol. Plat. Microb. Inter. 2011.24:163–171). Magnaporthe grisea expresses several heme peroxidases and catalases of remarkable structural and functional variability.In this work we have focused on heme b containing catalase-peroxidases (KatGs). The fungus expresses an intracellularKatG most likely located in peroxisomes (MagKatG1) as wellas an extracellular variant that is secreted and might participatein host-pathogen interaction. Recombinant MagKatG1 and MagKatG2 have been expressed heterologously in E. coli and purified to homogeneity. We report UV–Vis and resonance Raman spectra of the ferric and ferrous forms as well as the standard reduction potential of the Fe(III)/ Fe(II) couple of both metalloproteins. Kinetic parameters of both catalase and peroxidase activities are reported probing several artificial one- and two electron donors at different pH. Both described KatGs differed slightly in their pH optima for both catalatic and peroxidatic reactions. For probing the peroxidatic reaction mode several (artificial) one and two electron donors (ascorbate, ABTS, 5-amino salicylic acid, L-DOPA, o-dianisidine, guaiacol, resorcinol, catechol, pyrogallol, luminol, tyrosine, tetramethylbenzidine and halides) were tested since the natural substrate is unknown. In addition ligand binding and reaction of the ferric enzymes with various hydrogen peroxide and peroxoacetic acid concentrations were tested by stopped-flow spectroscopy.Similarities and differences between these two KatGs and omologous heme peroxidases will be discussed and related to a putative role of KatG2 in plant-pathogen interaction.

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

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

2011 - Influence of the Covalent Heme−Protein Bonds on the RedoxThermodynamics of Human Myeloperoxidase [Articolo su rivista]
Battistuzzi, Gianantonio; J., Stampler; Bellei, Marzia; J., Vlasits; M., Soudi; P. G., Furtmüller; C., Obinger
abstract

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the twoelectron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its(electronic) structure and biophysical and chemical properties.Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants withdisrupted heme−protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. Itturns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positivereduction potential (E°′ = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. Bycontrast, upon disruption of the MPO−typical sulfonium ion linkage, the reduction potential is significantly lower (−0.182 V).The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronouncedeffects (E°′ = −0.094 V), whereas in the D94V variant (E°′ = −0.060 V), formation of the ferrous state is entropically disfavored.These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants.

2011 - Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase [Articolo su rivista]
J., Stampler; Bellei, Marzia; M., Soudi; C., Gruber; Battistuzzi, Gianantonio; P. G., Furtmüller; C., Obinger
abstract

In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionatesubstituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (-50 mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidationstates showed significant differences. Additionally, rates of ligand binding and oxidation of both one and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His-Asn-Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate bindingsite(s) at the entrance to the heme cavity is discussed.

2011 - Redox Chemistry of the Schizosaccharomyces pombe Ferredoxin Electron-Transfer Domain and Influence of Cys to Ser Substitutions [Articolo su rivista]
S. P., Wu; Bellei, Marzia; S. S., Mansy; Battistuzzi, Gianantonio; Sola, Marco; J. A., Cowan
abstract

Schizosaccharomyces pombe (Sp) ferredoxin contains a C-terminal electron transfer protein ferredoxin domain(etpFd) that is homologous to adrenodoxin. The ferredoxin has been characterized by spectroelectrochemicalmethods, and Mössbauer, UV–Vis and circular dichroism spectroscopies. The Mössbauer spectrum isconsistent with a standard diferric [2Fe–2S]2+ cluster. While showing sequence homology to vertebrateferredoxins, the E°' and the reduction thermodynamics for etpFd (−0.392 V) are similar to plant-typeferredoxins. Relatively stable Cys to Ser derivatives were made for each of the four bound Cys residues andvariations in the visible spectrum in the 380–450 nm range were observed that are characteristic of oxygenligated clusters, including members of the [2Fe–2S] cluster IscU/ISU scaffold proteins. Circular dichroismspectra were similar and consistent with no significant structural change accompanying these mutations. Allderivatives were active in an NADPH-Fd reductase cytochrome c assay. The binding affinity of Fd to thereductase was similar, however, Vmax reflecting rate limiting electron transfer was found to decrease ~13-fold.The data are consistent with relatively minor perturbations of both the electronic properties of the clusterfollowing substitution of the Fe-bond S atom with O, and the electronic coupling of the cluster to the protein.

2010 - Control of Reduction Thermodynamics in [2Fe-2S] Ferredoxins. Entropy-Enthalpy Compensation and the Influence of Surface Mutations [Articolo su rivista]
Bellei, Marzia; Battistuzzi, Gianantonio; S. P., Wu; S. S., Mansy; J. A., Cowan; Sola, Marco
abstract

The reaction thermodynamics for the one-electron reduction of the [2Fe–2S] cluster of both human ferredoxin and various surface point mutants, in which each of the negatively charged residues Asp72, Glu73, Asp76, and Asp79 were converted to Ala, have been determined by variable temperature spectroelectrochemical measurements. The above are conserved residues that have been implicated in interactions between the vertebrate-type ferredoxins and their redox partners. In all cases, and similar to other 2Feferredoxins, the reduction potentials are negative as a result of both an enthalpic and entropic stabilization of the oxidized state. Although all Hs Fd mutants, with the exception of Asp72Ala, show slightly higher E°′values than that of wild type Hs Fd, according to expectations for a purely electrostatic model, they exhibit changes in the ΔH°′rc values that are electrostatically counter-intuitive. The observation of enthalpy–entropy compensation within the protein series indicates that the mutation-induced changes in ΔH°′rc and ΔS°′rc are dominated by reduction-induced solvent reorganization effects. Protein-based entropic effects are likely to be responsible for the low E°′ value of D72A.

2010 - Disruption of the H-bond network in the main access channel of catalase–peroxidasemodulates enthalpy and entropy of Fe(III) reduction [Articolo su rivista]
J., Vlasits; Bellei, Marzia; C., Jakopitsch; De Rienzo, Francesca; P. G., Furtmüller; M., Zamocky; Sola, Marco; Battistuzzi, Gianantonio; C., Obinger
abstract

Catalase–peroxidases are the only heme peroxidases with substantial hydrogen peroxide dismutation 28activity. In order to understand the role of the redox chemistry in their bifunctional activity, catalatically- 29active and inactive mutant proteins have been probed in spectroelectrochemical experiments. In detail, wild- 30type KatG from Synechocystis has been compared with variants with (i) disrupted KatG-typical adduct 31(Trp122-Tyr249-Met275), (ii) mutation of the catalytic distal His123-Arg119 pair, and (iii) altered 32accessibility to the heme cavity (Asp152, Ser335) and modified charge at the substrate channel entrance 33(Glu253). A valuable insight into the mechanism of reduction potential (E°′) modulation in KatG has been 34obtained from the parameterization of the corresponding enthalpic and entropic components, determined 35from the analysis of the temperature dependence of E°′. Moreover, model structures of ferric and ferrous 36Synechocystis KatG have been computed and used as reference to analyze and discuss the experimental data. 37The results, discussed by reference to published resonance Raman data on the strength of the proximal iron- 38imidazole bond and catalytic properties, demonstrate that E°′ of the Fe(III)/Fe(II) couple is not strongly 39correlated with the bifunctional activity. Besides the importance of an intact Trp-Tyr-Met adduct, it is the 40architecture of the long and constricted main channel that distinguishes KatGs from monofunctional 41peroxidases. An ordered matrix of oriented water dipoles is important for H2O2 oxidation. Its disruption 42results in modification of enthalpic and entropic contributions to E°′ that reflect reduction-induced changes 43in polarity, electrostatics, continuity and accessibility of solvent to the metal center as well as alterations in 44solvent reorganization.

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 - Redox properties of heme peroxidases [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Bortolotti, Carlo Augusto; Sola, Marco
abstract

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction 24of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and 25inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, 26involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of 27the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the 28reduction potentials of three redox couples, namely Compound I/Fe3+, Compound I/Compound II and 29Compound II/Fe3+. In particular, the oxidative power of heme peroxidases is controlled by the (high) 30reduction potential of the latter two couples. Moreover, the rapid H2O2-mediated two-electron oxidation 31of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on 32the reduction potential of the Fe3+/Fe2+ couple. The understanding of the molecular determinants of the 33reduction potentials of the above redox couples is crucial for the comprehension of the molecular deter- 34minants of the catalytic properties of heme peroxidases. 35This review provides an overview of the data available on the redox properties of Fe3+/Fe2+, Compound 36I/Fe3+, Compound I/Compound II and Compound II/Fe3+ couples in native and mutated heme peroxidases. 37The influence of the electron donor properties of the axial histidine and of the polarity of the heme envi- 38ronment is analyzed and the correlation between the redox properties of the heme group with the cat- 39alytic activity of this important class of metallo-enzymes is discussed

2010 - Redox thermodynamics of lactoperoxidase and eosinophil peroxidase [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; J., Vlasits; S., Banerjee; P. G., Furtmüller; Sola, Marco; C., Obinger
abstract

Eosinophil peroxidase (EPO) and lactoperoxidase (LPO) are important constituents of the innate immunesystem of mammals. These heme enzymes belong to the peroxidase-cyclooxygenase superfamily and catalyzethe oxidation of thiocyanate, bromide and nitrite to hypothiocyanate, hypobromous acid and nitrogendioxide that are toxic for invading pathogens. In order to gain a better understanding of the observeddifferences in substrate specificity and oxidation capacity in relation to heme and protein structure, acomprehensive spectro-electrochemical investigation was performed. The reduction potential (E0) ofthe Fe(III)/Fe(II) couple of EPO and LPO was determined to be 126 mV and 176 mV, respectively(25 C, pH 7.0). Variable temperature experiments show that EPO and LPO feature different reductionthermodynamics. In particular, reduction of ferric EPO is enthalpically and entropically disfavored,whereas in LPO the entropic term, which selectively stabilizes the oxidized form, prevails on the enthalpicterm that favors reduction of Fe(III). The data are discussed with respect to the architecture of theheme cavity and the substrate channel. Comparison with published data for myeloperoxidase demonstratesthe effect of heme to protein linkages and heme distortion on the redox chemistry of mammalianperoxidases and in consequence on the enzymatic properties of these physiologically importantoxidoreductases.

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 - 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 - Intracellular catalase/peroxidase from the phytopathogenic rice blast fungus Magnaporthe grisea: expression analysis and biochemical characterization of the recombinant protein [Articolo su rivista]
M., Zamocky; P. G., Furtmuller; Bellei, Marzia; Battistuzzi, Gianantonio; J., Stadlmann; J., Vlasits; C., Obinger
abstract

Phytopathogenic fungi such as the rice blast fungus Magnaporthegrisea are unique in having two catalase/peroxidase (KatG)paralogues located either intracellularly (KatG1) or extracellularly(KatG2). The coding genes have recently been shownto derive from a lateral gene transfer from a (proteo)bacterialgenome followed by gene duplication and diversification. Here wedemonstrate thatKatG1 is expressed constitutively in M. grisea. Itis the first eukaryotic catalase/peroxidase to be expressed heterologouslyin Escherichia coli in high amounts, with high purity andwith almost 100% haem occupancy. Recombinant MagKatG1is an acidic, mainly homodimeric, oxidoreductase with a predominantfive-co-ordinated high-spin haem b. At 25◦C andpH 7.0, the E0 (standard reduction potential) of the Fe(III)/Fe(II)couple was found to be −186+−10 mV. It bound cyanidemonophasically with an apparent bimolecular rate constant of(9.0+−0.4)×105 M−1 · s−1 at pH 7.0 and at 25◦C and with aKd value of 1.5 μM. Its predominantly catalase activity wascharacterized by a pH optimum at 6.0 and kcat and Km valuesof 7010 s−1 and 4.8 mM respectively. In addition, it acts as aversatile peroxidase with a pH optimum in the range 5.0–5.5using both one-electron [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) o-dianisidine, pyrogallol or guaiacol] andtwo-electron (Br−, I− or ethanol) donors. Structure–functionrelationships are discussed with respect to data reported forprokaryotic KatGs, as is the physiological role of MagKatG1.Phylogenetic analysis suggests that (intracellular) MagKatG1 canbe regarded as a typical representative for catalase/peroxidase ofboth phytopathogenic and saprotrophic fungi.

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 - Disruption of the aspartate to heme ester linkage in human myeloperoxidase: Impact on ligand binding, redox chemistry and interconversion of redox intermediates [Articolo su rivista]
M., Zederbauer; P. G., Furtmller; Bellei, Marzia; J., Stampler; C., Jakopitsch; Battistuzzi, Gianantonio; N., Moguilevsky; C., Obinger
abstract

In human heme peroxidases the prosthetic group is covalentlyattached to the protein via two ester linkages between conservedglutamate and aspartate residues and modified methyl groupson pyrrole rings A and C. Here, monomeric recombinantmyeloperoxidase (MPO) and the variants D94V and D94N wereproduced in Chinese hamster ovary cell lines. Disruption of theAsp94 to heme ester bond decreased the one-electron reductionpotential E0 [Fe(III)/Fe(II)] from 1 to 55 mV at pH 7.0 and25 °C, whereas the kinetics of binding of low spin ligands and ofcompound I formation was unaffected. By contrast, in both variantsrates of compound I reduction by chloride and bromide(but not iodide and thiocyanate) were substantially decreasedcompared with the wild-type protein. Bimolecular rates of compoundII (but not compound I) reduction by ascorbate and tyrosinewere slightly diminished in D94V and D94N. The presentedbiochemical and biophysical data suggest that the Asp94 to hemelinkage is no precondition for the autocatalytic formation of theother two covalent links found in MPO. The findings are discussedwith respect to the known active site structure of MPOand its complexes with ligands.

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 - Impact of heme to protein linkages in peroxidases on redox chemistry and catalysis [Abstract in Rivista]
C., Obinger; J., Vlasits; Bellei, Marzia; Battistuzzi, Gianantonio; P. G., Furtmüller
abstract

The mammalian peroxidases participate in host defence against infection, hormone synthesis and pathogenesis. The most striking feature of these heme enzymes is the existence of two covalent ester bonds between the prosthetic group and the protein in the functional, mature proteins. Myeloperoxidase is unique in having an additional vinyl-sulfonium bond. The impact of heme distortion and asymmetry on the spectral and enzymatic properties is discussed as is the role of the MPO-typical electron withdrawing sulfonium ion linkage in raising the reduction potential of its redox intermediatesand maintaining a rigid solvent network at the distal heme cavity.

2007 - Redox reactivity of the heme Fe3+/Fe2+ couple in native myoglobins and mutants with peroxidase-like activity [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; L., Casella; Bortolotti, Carlo Augusto; E., Monzani; R., Roncone; Sola, Marco
abstract

The reaction enthalpy and entropy for the one-electron reduction of the ferric heme in horse heart and sperm whale aquometmyoglobins (Mb) have been determined exploiting a spectroelectrochemical approach. Also investigated were the T67R, T67K, T67R/S92D and T67R/S92D Mb-H variants (the latter containing a protoheme-l</Emphasis>-histidine methyl ester) of sperm whale Mb, which feature peroxidase-like activity. The reduction potential (E°′) in all species consists of an enthalpic term which disfavors Fe3+ reduction and a larger entropic contribution which instead selectively stabilizes the reduced form. This behavior differs from that of the heme redox enzymes and electron transport proteins investigated so far. The reduction thermodynamics in the series of sperm whale Mb variants show an almost perfect enthalpy-entropy compensation, indicating that the mutation-induced changes in <EquationSource Format="TEX"><![CDATA[$$ Delta H^{{{^circ }ifmmode{'}else$'$fi }}_{{{text{rc}}}} ;{text{and }}Delta S^{{{^circ }ifmmode{'}else$'$fi }}_{{{text{rc}}}} {text{ }} $$]]></EquationSource> are dominated by reduction-induced solvent reorganization effects. The modest changes in E°′ originate from the enthalpic effects of the electrostatic interactions of the heme with the engineered charged residues. The small influence that the mutations exert on the reduction potential of myoglobin suggests that the increased peroxidase activity of the variants is not related to changes in the redox reactivity of the heme iron, but are likely related to a more favored substrate orientation within the distal heme cavity.

2007 - Redox thermodynamics of the Fe3+/Fe2+ couple in wild type and mutated heme peroxidases [Abstract in Rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; C., Jakopitsch; J., Vlasits; P. G., Furtmüller; Sola, Marco; C., Obinger
abstract

The thermodynamics of the one-electron reduction of the ferricheme in wild-type and mutated heme Synechocystis catalaseperoxidase and human myeloperoxidase were determined through spectro-electrochemical experiments. The data are interpreted in terms of ligand binding features, electrostatic effects and solvation properties of the heme environment.

2007 - Thermodynamics of the alkaline transition in phytocyanins [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; C., Dennison; DI ROCCO, Giulia; K., Sato; Sola, Marco; S., Yanagisawa
abstract

The thermodynamics of the alkaline transitionwhich influences the spectral and redox properties of thetype 1 copper center in phytocyanins has been determinedspectroscopically. The proteins investigated include Rhusvernicifera stellacyanin, cucumber basic protein and itsMet89Gln variant, and umecyanin, the stellacyanin fromhorseradish roots, along with its Gln95Met variant. Thechanges in reaction enthalpy and entropy within the proteinseries show partial compensatory behavior. Thus, thereaction free energy change (hence the pKa value) is rathervariable. This indicates that species-dependent differencesin reaction thermodynamics, although containing animportant contribution from changes in the hydrogenbondingnetwork of water molecules in the hydrationsphere of the protein (which feature enthalpy–entropycompensation), are to a large extent protein-based. Thedata for axial ligand variants are consistent with thehypothesis of a copper-binding His as the deprotonatingresidue responsible for this transition.

2006 - Redox properties of the Fe3+/Fe2+ couple in Arthromyces ramosus class II peroxidase and its cyanide adduct [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; DE RIENZO, Francesca; Sola, Marco
abstract

The thermodynamics of the one-electron reduction of the ferric heme in free and cyanide-bound Arthromyces ramosus peroxidase (ARP), a class II plant peroxidase, were determined through spectro-electrochemical experiments. The data were compared with those for class III horseradish peroxidase C (HRP) and its cyanide adduct, and were interpreted in terms of ligand binding features, electrostatic effects and solvent accessible surface area of the heme group and of catalytically relevant residues in the heme distal site. The E-o' values for free and cyanide-bound ARP (-0.183 and -0.390 V, respectively, at 25 degrees C and pH 7) are higher than those for HRP and HRP-CN. ARP features an enthalpic stabilization of the ferrous state and a remarkably negative reduction entropy, which are both unprecedented for heme peroxidases. Once the compensatory contributions of solvent reorganization are partitioned from the measured reduction enthalpy, the resulting protein-based Delta H-rc(int)degrees' value for ARP turns out to be less positive than that for HRP by + 10 kJ mol(-1). The smaller stabilization of the oxidized heme in ARP most probably results from the less pronounced anionic character of the proximal histidine, and the decreased polarity in the heme distal site as compared with HRP, as indicated by the X-ray structures. The surprisingly negative Delta S-rc degrees' value for ARP is the result of peculiar reduction-induced solvent reorganization effects.

2006 - Redox thermodynamics of the Fe(III)/Fe(II) couple of human myeloperoxidase in its high-spin and low-spin forms [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Zederbauer, M; Furtmuller, Pg; Sola, Marco; Obinger, C.
abstract

Myeloperoxidase (MPO) (donor, hydrogen peroxide oxidoreductase, EC 1.11.1.7) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride (Cl-), to generate the potent bleaching oxidant hypochlorous acid (HOCl), thus contributing to bacterial killing and inflammatory reactions of neutrophils. Here, the thermodynamics of the one-electron reduction of the ferric heme in its ferric high-spin and cyanide-bound low-spin forms were determined through spectroelectrochemical experiments. The E degrees' values for free and cyanide-bound MPO (5 and -37 mV, respectively, at 25 degrees C and pH 7.0) are significantly higher than those of other heme peroxidases. Variable-temperature experiments revealed that the enthalpic stabilization of ferric high-spin MPO is much weaker than in other heme peroxidases and is exactly compensated by the entropic change upon reduction. In contrast to those of other heme peroxidases, the stabilization of the ferric cyanide-bound MPO is also very weak and fully entropic. This peculiar behavior is discussed with respect to the MPO-typical covalent heme to protein linkages as well as to the published structures of ferric MPO and its cyanide complex and the recently published structure of lactoperoxidase as well as the physiological role of MPO in bacterial killing.

2006 - Redox thermodynamics of the ferric-ferrous couple of wild-type Synechocystis KatG and KatG(Y249F) [Articolo su rivista]
Bellei, Marzia; C., Jakopitsch; Battistuzzi, Gianantonio; Sola, Marco; C., Obinger
abstract

Crystal structures and mass spectrometric analyses of catalase-peroxidases (KatGs) from different organisms revealed the existence of a peculiar distal Met-Tyr-Trp cross-link. The adduct appears to be important for the catalase but not the peroxidase activity of bifunctional KatG. To examine the effect of the adduct on enzyme redox properties and functions, we have determined the thermodynamics of ferric reduction for wild-type KatG and KatG(Y249F), whose tyrosine-to-phenylalanine mutation prevents cross-link formation. At 25 degrees C and pH 7.0, the reduction potential of wild-type KatG is found to be -226 +/- 10 mV, remarkably lower than the published literature values. The reduction potential of KatG(Y249F) is very similar (-222 +/- 10 mV), but variable temperature experiments revealed compensatory differences in reduction enthalpies and entropies. In both proteins, the oxidized state is enthalpically stabilized over the reduced state, but entropy is lost on reduction, which is in strong contrast to horseradish peroxidase, which also features a much more pronounced enthalpic stabilization of the ferriheme. With both proteins, the midpoint potential increased linearly with decreasing pH. We discuss whether the observed redox thermodynamics reflects the differences in structure and function between bifunctional KatG and monofunctional peroxidases.

2006 - Structural features in EIAV NCp11: A lentivirus nucleocapsid protein with a short linker [Articolo su rivista]
Amodeo, P; Morelli, Mac; Ostuni, A; Battistuzzi, Gianantonio; Bavoso, A.
abstract

Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential role in RNA packaging and viral infectivity. They contain two CX2CX4HX4C zinc binding motifs connected by a basic linker of variable length. The 3D structure of a 37-aa peptide corresponding to sequence 2258 from lentiviral EIAV nucleocapsid protein NCp11, complexed with zinc, has been determined by 2D H-1 NMR spectroscopy, simulated annealing, and molecular dynamics. The solution structure consists of two zinc binding domains held together by a five-residue basic linker Arg(38)-Ala-Pro-Lys-Val(42) that allows for spatial proximity between the two finger domains. Observed linker folding is stabilized by H bonded secondary structure elements, resulting in an Q-shaped central region, asymmetrically centered on the linker. The conformational differences and similarities with other NC zinc binding knuckles have been systematically analyzed. The two CCHC motifs, both characterized by a peculiar Pro-Gly sequence preceding the His residue, although preserving Zn-binding geometry and chirality of other known NC proteins, exhibit local fold differences both between each other and in comparison with other previously characterized retroviral CCHC motifs.

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

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

2005 - Acqua: questa meravigliosa sconoscita [Esposizione]
Brunetti, Rossella; Battistuzzi, Gianantonio
abstract

PROMOTORIBiblioteca Civica Delfini e Biblioteca Scientifica Interdipartimentale (BSI) della Università di Modena e Reggio EmiliaDESTINATARIScuole superiori, quarte e quinte classiDESCRIZIONE E FINALITA’L'acqua è il composto chimico più conosciuto, quello con cui si ha a che fare ogni giorno, che non può mancare nella nostra vita. L'unico di cui quasi tutti conoscono la formula chimica. Ma pochi sanno che l’acqua ha proprietà chimiche e fisiche diverse da quelle degli altri liquidi. Proprietà che la rendono speciale per ospitare e favorire la vita, e ne fanno un meraviglioso solvente utilizzato per mille usi. L'acqua è un bene prezioso. Capace, da sempre, di scatenare sanguinosi conflitti. La Terra è l’unico oggetto cosmico conosciuto dove questa bizzarra molecola chimica non solo è presente in grande massa, ma anche in tutti e tre gli stati di aggregazione: solido, liquido e gassoso.Il progetto pluriennale La curiosità fa lo scienziato. Leggere e condividere la scienza di oggi in ciascuna edizione approfondisce un tema scientifico di rilievo, impegnando le classi aderenti (monitorate da un esperto) per l’intero anno scolastico. Lo scopo è avvicinare i ragazzi al mondo della ricerca, formare interessi e capacità critiche nei confronti della letteratura di divulgazione e, più in generale, dei codici di comunicazione della scienza contemporanea. Inoltre, promuovere un ruolo attivo dei giovani nella disseminazione delle conoscenze scientifiche: impegnati nella stesura di un breve articolo, gli studenti mettono alla prova la loro capacità di spiegare in modo chiaro e conciso concetti a volte complessi.Quest’anno le classi partecipanti lavoreranno su testi divulgativi sulle proprietà chimiche dell’acqua e sulle loro implicazioni (fisiche, biologiche, geologiche, sociali, antropologiche, ecologiche), con possibili incursioni su aspetti meno noti trattati nei libri suggeriti.REFERENTI•Rossella Brunetti (BSI e Dipartimento di Fisica, UniMoRe, tel 059 2055277)•Gianantonio Battistuzzi, Monica Saladini (Dipartimento di Chimica, UniMoRe, tel 059 2055117)•Cinzia Pollicelli, Angela Pacillo (Servizio Biblioteche, tel 059 2032798)PRENOTAZIONEIl progetto si rivolge alle classi quarte e quinte. E' a numero chiuso con prenotazione obbligatoria (massimo 5 classi). Si raccomanda un contatto telefonico preliminare con uno dei referenti.www.comune.modena.it/istruzione/itinerariCALENDARIO DI LAVOROGiovedì 20 ottobre 2005, ore 17Incontro tra organizzatori, insegnanti e alcuni rappresentanti degli studenti: informazioni preliminari e coordinamento sull'iter del progetto.Biblioteca Scientifica Interdipartimentale, Via Campi 213/C, ModenaLunedì 14 novembre 2005, ore 10,30Esplorazione scientifica del pianeta acqua, lezione introduttiva del Prof. GIANANTONIO BATTISTUZZI (UniMoRe, Dip. di Chimica); presentazione delle letture proposte agli studenti e distribuzione dei libri.Biblioteca Scientifica Interdipartimentale, Via Campi 213/C, ModenaNovembre 2005-Marzo 2006Laboratorio in classe, guidato dagli insegnanti di chimica, scienze e lettere, a partire dai volumi distribuiti e dagli spunti offerti dalla lezione introduttiva. Su modello del giornalismo scientifico, gli studenti avviano la redazione di un articolo sul tema dell’acqua, da completare con il successivo incontro-intervista con un esperto della materia. Preparazione di un elenco di domande da rivolgere all'esperto. Durante questa fase di lettura ed elaborazione, l'Università di Modena e Reggio Emilia offre consulenza scientifica a insegnanti e studenti su problemi specifici.Marzo 2006, data da definire, ore 10,30Incontro-intervista con un esperto che illustra la propria biografia scientifica e risponde alle domande dei ragazzi.Biblioteca civica Delfini, corso Canalgrande 103, ModenaMarzo 2006, data da definire, ore 21Conferenza serale sul tema dell’acqua aperta alla cittadinanza.Biblioteca Scientifica Interdipartimentale, Via Campi 213/C, ModenaAprile

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

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

2005 - Reduction thermodynamics of the T1 Cu site in plant and fungal laccases [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Leonardi, Alan; R., Pierattelli; Aj, Vila; A., DE CANDIA; Sola, Marco
abstract

The thermodynamic parameters for reduction of the type-1 (T1) copper site in Rhus vernicifera and Trametes versicolor laccases and for the derivative of the former protein from which the type-2 copper has been selectively removed (T2D) have been determined with UV-vis spectroelectrochemistry. In all cases, the enthalpic term turns out to be the main determinant of the E-o' of the T1 site. Also the difference between the reduction potentials of the two laccases is enthalpy-based and reflects differences in the coordination features of the T1 sites and their protein environment. The T1 sites in native R. vernicifera laccase and its T2D derivative show the same E-o', as a result of compensatory differences in the reduction thermodynamics. This suggests that removal of the type-2 (T2) copper results in modification of the reduction-induced solvent reorganization effects, with no influence in the structure of the multicopper protein site. This conclusion is supported by NMR data recorded on the native, the T2D, and Hg-substituted T1 derivatives of R. vernicifera laccase, which show that the T1 and T2/T3 sites are largely noninteracting.

2004 - Characterization of the solution reactivity of a basic heme peroxidase from Cucumis sativus [Articolo su rivista]
Battistuzzi, Gianantonio; Bellei, Marzia; Bortolotti, Carlo Augusto; DI ROCCO, Giulia; Leonardi, Alan; Sola, Marco
abstract

A basic heme peroxidase has been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and H NMR spectra from pH 3 to 11. The protein, as isolated, contains a high-spin ferriheme which in the low pH region is sensitive to two acid-base equilibria with apparent pK(a) values of approximately 5 and 3.6, assigned to the distal histidine and to a heme propionate, respectively. At high pH, a new low-spin species develops with an apparent pK(a) of 11, likely due to the binding of an hydroxide ion to the sixth (axial) coordination position of the Fe(III). A number of acid-base equilibria involving heme propionates and residues in the distal cavity also affect the binding of inorganic anions such as cyanide, azide, and fluoride to the ferriheme, as well as the catalytic activity. The reduction potentials of the native protein and of its cyanide derivative, determined through UV-Vis spectroelectrochemistry, result to be -0.320 +/- 0.015 and -0.412 +/- 0.010V, respectively. Overall, the reactivity of this protein parallels those of other plant peroxidases, especially horseradish peroxidase. However, some differences exist in the acid-base equilibria affecting its reactivity and in the reduction potential, likely as a result of small structural differences in the heme distal and proximal cavities.

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 - Grabbing Yeast iso-1-cytochrome c by Cys102: an effective approach for the assembly of functionally active metalloprotein carpets [Articolo su rivista]
Gerunda, Mimmo; Bortolotti, Carlo Augusto; Alessandrini, Andrea; Sola, Marco; Battistuzzi, Gianantonio; P., Facci
abstract

We report an approach for immobilizing iso-l-cytochrome c from Saccharomyces cerevisiae on oxygen exposing surfaces derivatized with SH-terminated silanes. The SH moieties from silanes have been brought to react with the partially buried Cys102, forming an intermolecular disulfide bond which anchored covalently cytochrome c to the surface. The presence of a single cysteine residue on the protein surface imparted a well-defined orientation to the molecular edifice. Molecular constructs obtained with native cytochrome c and with a cysteine-depleted mutant (C102T) have been investigated by means of scanning force microscopy under liquid, which was performed to assay the quality of the molecular carpet, showing that the native protein formed a robust monolayer at the surface, whereas only a negligible amount of physisorbed molecules were detected in the case of a mutant. UV-vis absorption spectroscopy was performed to confirm that immobilization takes place via the Cys102 residue. Linear sweep voltammetric measurements showed retention of the redox activity of the covalently immobilized cytochrome c, confirming the viability of the proposed immobilization method for obtaining monolayers of redox active molecules.

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 - 1H NMR of native and azide-inhibited laccase from Rhus vernicifera [Articolo su rivista]
Battistuzzi, Gianantonio; DI ROCCO, Giulia; Leonardi, Alan; Sola, Marco
abstract

The H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time.These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in thehigh frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differencesbetween the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structuralproperties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over1 the T1 copper ligands. Overall, these data demonstrate that H NMR can be fruitfully applied to characterize the electronic properties ofthe metal sites of blue oxidases at room temperature.

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 - 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 - Metal-Ligand Interplay in Blue Copper Proteins Studied by 1H NMR Spectroscopy: Cu(II)-Pseudoazurin and Cu(II)-Rusticyanin. [Articolo su rivista]
BATTISTUZZI, Gianantonio; SOLA, Marco
abstract

In this work, the authors have extended the 1H NMR analysis to the “perturbed” tetragonal blue copper centers of the oxidized forms of Achromobacter cycloclastes pseudoazurin and Thiobacillus ferrooxidans rusticyanin (Fig. 1).New important information is provided on the molecular factors that control the electronic properties of these important metal sites.

2002 - A Comparison of the Inner-Sphere Reorganization Energies of Cytochromes, Iron-Sulfur Clusters and Blue Copper Proteins. [Articolo su rivista]
Battistuzzi, Gianantonio; Sola, Marco
abstract

Abstract:Inner-sphere reorganization energies have been calculated for a number of models of six-coordinate iron porphyrins (with varying axial ligands), using the density functional B3LYP method. If the axial ligands are uncharged, the reorganization energy is very low, 5-9 kJ/mol. If one of the axial ligands is charged, the reorganization energy is higher, 20-47 kJ/mol, but such sites are normally not used in electron carriers. The former reorganization energies are appreciably smaller than what was found for blue copper proteins (62-90 kJ/mol), the dimeric CuA site in cytochrome c oxidase and nitrous oxide reductase (43 kJ/mol), and six different types of iron-sulfur clusters with one, two, or four iron atoms (40-75 kJ/mol), even if these vacuum energies are typically halved inside the protein (as a result of hydrogen bonds and solvation effects). Therefore, the cytochromes seem to have the inherently lowest inner-sphere reorganization energy of the three commonly used electron carriers. All three types of sites have reduced the reorganization energy by using a delocalized charge and N- and S-donors (rather than O-donors) as metal ligands. Moreover, iron is a more appropriate metal for electron transfer than copper because Fe(II) and Fe(III) prefer the same coordination number and geometry and give bonds weaker than those of copper. The low-spin state of the cytochrome has a ~20 kJ/mol lower reorganization energy than that of the corresponding high-spin site. Moreover, ring strain in the porphyrin reduce the changes in the Fe-NPor distances by 5 pm and therefore the reorganization energy by 8 kJ/mol.

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

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

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

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

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

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

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

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

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

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

2001 - Isolation and characterization of two peroxidases from Cucumis sativus [Articolo su rivista]
Battistuzzi, Gianantonio; M., D'Onofrio; Loschi, Lodovica; Sola, Marco
abstract

Two heme peroxidases of 35.2 and 38.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and IH NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture elf two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pK(a) values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; how ever, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.

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

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

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

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

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

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

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

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

2001 - Synthesis, crystal and molecular structure, spectroscopic and electrochemical studies of trichloro-oxo(4,6-dimethypyrimidine-2(1H)-thione)(triphenylphosphine oxide) rhenium(V) complex. [Articolo su rivista]
Battistuzzi, Gianantonio; Cannio, Maria; Saladini, Monica; Battistuzzi, Raffaele
abstract

Reaction of 4,6-dimethylpyrimidine-2(1H)-thione (Me2pymSH) with mer-[ReOCl3(Me2S)(OPPh3)] synthon in 1:1 molar ratio inrefluxing acetone, results in the replacement of the Me2S ligand to form the mer-[ReOCl3(Me2pymSH)(OPPh3)] species. X-raydiffraction shows that the structure of the title compound consists of monomeric units with a distorted octahedral coordinationaround the rhenium(V) centre which includes the axial ReO and ReOPPh3 bonds, and in which three Cl− ions and aS-monodentate neutral Me2pymSH ligand act as equatorial ligands. The compound was also characterised using electrochemicalmeasurements and UV–Vis–NIR and IR spectroscopy.

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

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

2000 - Synthesis, magnetic, spectroscopic and electrochemical studies of mixed pyrimidine-2-thiolate/triphenylphosphine rhenium(V) and rhenium(III) complexes [Articolo su rivista]
Battistuzzi, Gianantonio; Cannio, Maria; Battistuzzi, Raffaele
abstract

Equimolar amounts of trans-[ReOX2(OEt)(PPh3)2] (XCl, Br, I) precursors and potentially bidentate N,S-donor pyrimidine-2(1H)-thione (pymSH) react in refluxing acetone to give mononuclear octahedral paramagnetic trans-[ReIIIX2(pymS)(PPh3)2](XCl, Br, I) species. Starting from a metal–ligand molar ratio of 1:3, in the presence of N(C2H5)3 as deprotonating agent inrefluxing ethanol, the same reaction proceeds stepwise, affording octahedral [ReO(pymS)3] or [ReO(pymS)3] and pentagonalbipyramidal[Re(pymS)3PPh3] complexes as a function of the reaction time. The compounds were characterized by elementalanalysis, magnetic susceptibility, UV–Vis–NIR, IR and 1H NMR spectroscopy and by cyclovoltammetric measurements.Reaction pathways and physico-chemical properties of the complexes are discussed.

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

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

1999 - H-1 NMR of oxidized blue copper proteins [Articolo su rivista]
Battistuzzi, Gianantonio; Loschi, Lodovica; Sola, Marco
abstract

The hyperfine-shifted H-1 NMR resonances arising from the Cu(II) ligands of cucumber stellacyanin, plantacyanin (CBP) and plastocyanin, horseradish umecyanin, and of spinach plastocyanin and its L12G mutant are reported. It is shown that these broad paramagnetic signals are diagnostic of structural, electronic and solvation properties of the metal site and can be exploited to probe for differences and/or analogies among type I copper centers. (C) 1999 Elsevier Science Inc. All rights reserved.

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

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

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

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

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

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

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

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

1998 - Metal ion binding to a zinc finger peptide containing the Cys-X-2-Cys-X-4-His-X-4-Cys domain of a nucleic acid binding protein encoded by the Drosophila Fw-element [Articolo su rivista]
A., Bavoso; A., Ostuni; Battistuzzi, Gianantonio; Menabue, Ledi; Saladini, Monica; Sola, Marco
abstract

The metal binding properties of a 18-residue zinc finger peptide containing a CCHC box which reproduces one of the cysteine-rich domains of a putative nucleic acid binding protein encoded by the Fw transposable element from Drosophila melanogaster were investigated through electronic and H-1 NMR spectroscopy. Dissociation constants of 2(+/-1)x10(-12) M and 4(+/-1)x10(-7) M were determined for the Zn2+ and Co2+ adduct, respectively. These values are similar to those for other CCHC-peptides investigated previously, although the length of the spacer between the second cysteine and the histidine apparently exerts some influence on the spectral properties and on the stability of the Co2+-peptide adduct. The 1H NMR spectrum of the present Co2+-derivative contains a number of well resolved hyperfine-shifted resonances between 350 and -50 ppm which arise from the metal binding residues and nearby groups. These peaks can in principle be profitably exploited to monitor protein-nucleic acid interactions.

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

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

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

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

1998 - Synthesis, Crystal and Molecular Structure, Spectroscopic and Electrochemical Studies of -oxo-bis{oxo-bis(4,6-dimethylpyrimidine-2-thiolate)rhenium(V)} Complex [Articolo su rivista]
Battistuzzi, Gianantonio; Corradi, Anna; D., Dallari; Saladini, Monica; Battistuzzi, Raffaele
abstract

The dinuclear oxo!rhenium"V#\ m!OReO"Me1pymS#11 species was prepared by the reaction of 3\5!dimethylpyrimidine!1"0H#!thione "Me1pymSH# with trans!ReI1"EtO# "PPh2#1 or trans!ReO1py3Cl=1H1O in di}erent solvents[ The compounds were char!acterized by elemental analysis\ magnetic susceptibility\ UV!VIS\ IR and 0H NMR spectroscopy and conductivity measurements[Cyclic voltammetry shows that the m!OReO"Me1pymS#11 species\ undergo a quasi!reversible single!electron process in which theoxygen!bridged dimeric framework is maintained[ The structure of m!OReO"Me1pymS#11 was determined by single!crystal X!raydi}raction methods[ The dinuclear structure of Re1O2"Me1pymS#3 compound consists of two ReO"Me1pymS#1 moieties bridgedby an oxygen atom[ The backbone O1ReORe1O is almost linear and each rhenium"V# ion lies in the center of a distortedoctahedron achieved through two deprotonated N\S!chelate ligands in the equatorial plane[ 0888 Elsevier Science Ltd[ All rightsreserved[

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

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

1997 - Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: H-1 NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean [Articolo su rivista]
Battistuzzi, Gianantonio; M., Dietrich; R., Locke; H., Witzel
abstract

The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (M-r 111 000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)/Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted H-1 NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

1995 - METAL-INDUCED CONFORMATIONAL HETEROGENEITY OF TRANSFERRINS - A SPECTROSCOPIC STUDY OF INDIUM(III) AND OTHER METAL(III)-SUBSTITUTED TRANSFERRINS [Articolo su rivista]
Battistuzzi, Gianantonio; Calzolai, L; Messori, L; Sola, Marco
abstract

The conformation in solution of three different metal(III)-transferrins, namely aluminum(III), gallium(III) and indium(III) transferrin, was investigated by absorption, CD, H-1 NMR and C-13 NMR spectroscopies. The formation of the respective metal-transferrin complexes and the characteristic 2:1 metal-to-protein binding stoichiometry were unambiguously demonstrated, in all cases, through UV difference studies. The C-13 NMR spectra of these metallotransferrins in the carbonyl region are very similar to one another pointing out that the arrangement of the synergistic anion in the binding site must be essentially the same. However, the CD spectra in the near UV (aromatic region) reveal the occurrence of significant differences between indium transferrin, on one side, and the other two derivatives, on the other. Also, the H-1 NMR spectra exhibit a number of different features suggesting the occurrence of metal-induced conformational heterogeneity around the metal sites. Such metal-induced conformational heterogeneity probably affects the transferrin-receptor recognition process, resulting in a different metabolic fate of these metals in the organisms.

1995 - POLYMETALLIC HYDROLYTIC ZINC ENZYMES - PROBING THE SITE OF NUCLEASE P1 THROUGH COBALT(II) SUBSTITUTION [Articolo su rivista]
Ferretti, S; Luchinat, C; Sola, Marco; Battistuzzi, Gianantonio
abstract

Partial Co(II) substitution in the three-zinc site of P. citrinum nuclease P1 has been achieved. The Co(II) ion is found to bind to the site of the most EDTA-labile Zn atom with an 80% site occupancy. An affinity constant of 10(5) M(-1) for metal binding was determined from the visible spectra which also indicate a six-coordinate Co(II) geometry. The hyperfine-shifted H-1 NMR resonances suggest that metal substitution occurred at the Zn3 site (X-ray atom numbering).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

1992 - FE-3+ BINDING TO OVOTRANSFERRIN IN THE PRESENCE OF ALPHA-AMINO-ACIDS [Articolo su rivista]
Battistuzzi, Gianantonio; Sola, Marco
abstract

The ability of L-alpha-amino acids as synergistic anions for iron binding to ovotransferrin was investigated through electronic spectroscopy. Glycine and glutamic acid were found to form by far the most stable ternary Fe3+-ovotransferrin-amino acid complexes. Less stable adducts were formed by amino acids with a hydroxy, amide or sulphur-containing group in the side chain, while the complexes with leucine, isoleucine, valine, lysine, arginine, tyrosine and tryptophan failed to form. Evidence is obtained that the synergistic effectiveness of the H2N-CH-COO- moiety is determined not only by the isoelectric point of the amino acid and the steric hindrance of its side chain, but a significant role is also played by interactions of the side chain itself with residues in the metal binding domains. Zn2+, Cd2+ and Co2+ are found to bind to ovotransferrin in the presence of glycine. Cd-113-NMR spectra on the Cd-derivative indicate that, according to the interlocking-sites model, the amino group of glycine directly binds to the metal ion.