Pedro Tavares

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.

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.

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N2O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies (TM)). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

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.

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.

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).

Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alpha H195Q, alpha H195N, and alpha Q191 K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N-2 with the alpha H195Q and alpha H195N variants, but not with the alpha Q191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alpha H195Q or alpha H195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alpha Q191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze Fe-57 Mossbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alpha H195Q and alpha H195N MoFe proteins can bind N-2, but alpha Q195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N-2 reduction. (C) 2007 Elsevier Inc. All rights reserved.

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.

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.

{Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K(3)Fe(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K(3)Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-)(1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNlr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe(3+) ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

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.

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.

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.

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.

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.

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein, Selenium was not detected. The UV/visible absorption spectrum of D, gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with Fe-57 and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

The soluble methane monooxygenase system from Methylococcus capsulatus (Bath) catalyzes the oxidation of methane to methanol and water utilizing dioxygen at a non-heme, carboxylate-bridged diiron center housed in the hydroxylase (H) component. To probe the nature of the reductive activation of dioxygen in this system, reactions of an analogous molecule, nitric oxide, with the diiron(II) form of the enzyme (H-red) Were investigated by both continuous and discontinuous kinetics methodologies using optical, EPR, and Mossbauer spectroscopy. Reaction of NO with H-red affords a dinitrosyl species, designated H-dinitrosyl, with optical spectra (lambda(max) = 450 and 620 nm) and Mossbauer parameters (delta = 0.72 mm/s, Delta E-Q = 1.55 mm/s) similar to those of synthetic dinitrosyl analogues and of the dinitrosyl adduct of the reduced ribonucleotide reductase R2 (RNR-R2) protein. The H-dinitrosyl species models features of the H-peroxo intermediate formed in the analogous dioxygen reaction. In the presence of protein B, H-dinitrosyl builds up with approximately the same rate constant as H-peroxo (similar to 26 s(-1)) at 4 degrees C. In the absence of protein B, the kinetics of H-dinitrosyl formation were best fit with a biphasic A –> B –> C model, indicating the presence of an intermediate species between H-red and H-dinitrosyl. This result contrasts with the reaction of H-red with dioxygen, in which the H-peroxo intermediate forms in measurable quantities only in the presence of protein B. These findings suggest that protein B may alter the positioning but not the availability of coordination sites on iron for exogenous ligand binding and reactivity.