Pedro Tavares

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe3+/Fe2+ couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and IT or oxyferrous KatG.

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mossbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe-B). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while FeB was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mossbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g similar to 6 and g similar to 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g similar to 6 region were assigned to a small quantity of uncoupled high-spin Fe-III heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin spin coupled binuclear center comprising the low-spin Fe-III heme b(3) and the high-spin Fe-B(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe-II-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe-B(II) site. The function of heme b(3) would then be to orient the Fe-B-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.

Denitrification, or dissimilative nitrate reduction, is an anaerobic process used by some bacteria for energy generation. This process is important in many aspects, but its environmental implications have been given particular relevance. Nitrate accumulation and release of nitrous oxide in the atmosphere due to excess use of fertilizers in agriculture are examples of two environmental problems where denitrification plays a central role. The reduction of nitrate to nitrogen gas is accomplished by four different types of metalloenzymes in four simple steps: nitrate is reduced to nitrite, then to nitric oxide, followed by the reduction to nitrous oxide and by a final reduction to dinitrogen. In this manuscript we present a concise updated review of the bioinorganic aspects of denitrification. (c) 2006 Elsevier Inc. All rights reserved.

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mössbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal at g = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximately 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mössbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mössbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S–>Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2 Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mössbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.

Reactive oxygen species (ROS), when in excess, are among the most deleterious species an organism can deal with. The physiological effects of ROS include amino acid chain cleavage, DNA degradation and lipid oxidation, among others. They can be formed in the cytoplasm in a variety of ways, including autooxidation reactions (FMN- and FAD-containing enzymes) and Fenton reactions as a result of the cytoplasmatic pool of iron ions. The superoxide anion (021, despite its short half-life in solution, is particularly pernicious as it can form other reactive ROS (such as the strong oxidant peroxynitrite) or oxidize and/or reduce cellular components. For strict anaerobic or microaerophilic bacteria it is of particular importance to be able to dispose of ROS in a controlled manner, especially if these organisms are temporarily exposed to air. This review aims to describe the structural characteristics of superoxide reductases (SORs) and mechanistic aspects of biological superoxide anion reduction. SORs can be considered the main class of enzymes behind the oxygen detoxification pathway of anaerobic and microaerophilic bacteria. The geometry of the active site (three classes have been described), the possible electron donors in vivo and the current hypothesis for the catalytic mechanism will be discussed. Some phylogenetic considerations are presented, regarding the primary structure of SORs currently available in genome databases. ((c) Wiley-VCH Verlag GmbH {&} Co. KGaA, 69451 Weinheim, Germany, 2007).

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms, At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified, These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the ''young core.'' The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+- tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., {&} Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem, J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of similar to 80 s(-1) and the remainder at a much slower rate of similar to 1.7 s(-1). In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (Delta E-Q = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (lambda(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O-2 With the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S]H) and-a binuclear Fe cluster ([2Fe]H), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O-2 indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mossbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the GO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters,and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S] - [2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S]H cluster from the 2+ to the If state, followed by transfer of the reducing equivalent from the [4Fe-4S]H subcluster to the binuclear [2Fe]H subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S]H and the [2Fe]H subclusters. Implication of such a CO binding effect is discussed.

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfide-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mossbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data. (C) 1999 Academic Press.

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using C-13-detection experiments.

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mossbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8 Angstrom resolution. The iron atoms in the native protein were used as the anomalous scatterers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9 Angstrom. Desulfoferrodoxin is a homodimer which can be described in terms of two domains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is similar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square pyramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarkable structural fit with the DX homodimer. Furthermore, three beta-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisation process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-terminal domain in the monomer has a topology fold similar to that of fibronectin III.

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F(peroxo), formed within 25 ms, which then leads to the release of H(2)O(2) and formation of ferric mineral precursors. The stoichiometric relationship between F(peroxo), H(2)O(2), and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H(2)O(2). By rapidly mixing apo M ferritin from frog, Fe(2+), and O(2) and allowing the reaction to proceed for 70 ms when F(peroxo) has reached its maximum accumulation, followed by spraying the reaction mixture into the H(2)O(2) assay solution, we were able to quantitatively determine the amount of H(2)O(2) produced during the decay of F(peroxo). The correlation between the amount of H(2)O(2) released with the amount of F(peroxo) accumulated at 70 ms determined by Mossbauer spectroscopy showed that F(peroxo) decays into H(2)O(2) with a stoichiometry of 1 F(peroxo):H(2)O(2). When the decay of F(peroxo) was monitored by rapid freeze-quench Mossbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F(peroxo). This observed parallel formation of multiple products (H(2)O(2), diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F(peroxo)) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence (129)Cys-X-5-His-X-15-Cys-X-2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain. (C) 2008 Elsevier Inc. All rights reserved.

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

Superoxide reductases are enzymes involved in bacterial resistance to reactive oxygen species, catalyzing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-center containing domains. Most studies have been focused on the catalytic iron site (center II), but the role of center I is yet poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetics measurements as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted center I, maintaining the iron active site center II was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8x107 (M-1s-1) and 1.3x106 (M-1s-1) for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2x106 (M-1s-1) for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that center I transfers electrons from the putative physiologic donor, rubredoxin, to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated to the redox state of center I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.