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Marzia BELLEI

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Chimica

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Pubblicazioni

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

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.

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 - 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 - 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 - 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.

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 - 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.

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 - 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.

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.

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 - 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 - 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 - 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.

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 - 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.

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 - 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.

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

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

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

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

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

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

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

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