Aldehyde oxidoreductase (AOR) activity has been found in a number of sulfate-reducing bacteria. The enzyme that is responsible for the conversion of aldehydes to carboxylic acids is a mononuclear molybdenum enzyme belonging to the xanthine oxidase family. We report here the purification and characterization of AOR isolated from the sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254, an aminolytic strain performing thiosulfate dismutation. The enzyme is a homodimer (ca. 200 kDa), containing a molybdenum centre and two [2Fe-2S] clusters per monomer. UV/Visible and electron paramagnetic resonance (EPR) spectra of D. aminophilus AOR recorded in as-prepared and reduced states are similar to those obtained in AORs from Desulfovibrio gigas, Desulfovibrio desulfuricans and Desulfovibrio alaskensis. Despite AOR from D. aminophilus is closely related to other AORs, it presents lower activity towards aldehydes and no activity towards N-heterocyclic compounds, which suggests another possible role for this enzyme in vivo. A comparison of the molecular and EPR properties of AORs from different Desulfovibrio species is also included.

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A(278nm) / A(388nm)) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c(3) (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A(553nm) - A(570nm)](red) / A(280nm)) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c(3) from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).

We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126+/-2 kDa composed of two subunits, alpha=93+/-3 kDa and beta=32+/-2 kDa, which contains 6+/-1 Fe/molecule, 0.4+/-0.1 Mo/molecule, 0.3+/-0.1 W/molecule, and 1.3+/-0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d(1) configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.

The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2)) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b(5) has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b(5).

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe2+ chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.

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.

We report on a new method to make nanostructures in aqueous solution at room temperature. We used the protein cytochrome c(3) to catalyze reduction of selenate (SeO42-) to selenium Se-0 by dithionite. Reduction was instantaneous. After a week spherical nanoparticles of red Se-0 (about 50 nm diameter) precipitated, followed by self-assembling into crystalline nanowires, typically 1 mu m long. The nanowires were composed of one strand of spherical particles; thicker strands contained several nanoparticles in parallel.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis-Menten kinetics, with the following parameters (for benzaldehyde): K(app/m)= 6.65 microM; V app = 13.12 microM.min(-1); k(app/cat) = 0.96 s(-1). Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe-2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 mu M and allowing a maximal catalytic centre activity of 116 000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All rights reserved.

A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10(-8) - 10(-2) M range with a detection limit of 10(-8) M. and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NIR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10(-5) - 10(-2) M) and a higher detection limit (10(-5) M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal-coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c3. NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (nu4, nu3, and nu2) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c3 (Norway 4 and 9974) than for the Desulfovibrio (D.) gigas, D. vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the nu3 and nu10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins.

A novel sulphate-reducing bacterium (Ind 1) was isolated from a biofilm removed from a severely corroded carbon steel structure in a marine environment. Light microscopy observations revealed that cells were Gram-negative, rod shaped and very motile. Partial 16S rRNA gene sequencing and analysis of the fatty acid profile demonstrated a strong similarity between the new species and members from the Desulfovibrio genus. This was confirmed by the results obtained following purification and characterisation of the key proteins involved in the sulphate-reduction pathway. Several metal-containing proteins, such as two periplasmic proteins: hydrogenase and cytochrome c3, and two cytoplasmic proteins: ferredoxin and sulphite reductase, were isolated and purified. The latter proved to be of the desulfoviridin type which is typical of the Desulfovibrio genus. The study of the remaining proteins revealed a high degree of similarity with the homologous proteins isolated from Desulfovibrio gigas. However, the position of the strain within the phylogenetic tree clearly indicates that the bacterium is closely related to Desulfovibrio gabonensis, and these three strains form a separate cluster in the delta subdivision of the Proteobacteria. On the basis of the results obtained, it is suggested that Ind 1 belongs to a new species of the genus Desulfovibrio, and the name Desulfovibrio indonensis is proposed.

The periplasmic [NiFe] hydrogenase isolated from Desulfovibrio (D.) desulfuricans (ATCC 27774) is a heterodimer of a 28 kDa (beta) and a 60 kDa (alpha) subunit. Here we report the complete amino acid sequence of the small (beta) polypeptide chain determined by Edman degradation of proteolytic fragments. Electrospray-ionization mass spectrometry of the native protein confirmed the sequencing results. The sequence is compared with that of D. gigas [NiFe] hydrogenase whose three-dimensional structure has been recently published.

The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S](2+) cluster and is EPR-silent. The 8 T Mossbauer spectrum of the Fe-57-enriched as-isolated protein is well simulated by parameters Delta E(Q) = 0.69 +/- 0.03 mm/s and delta = 0.28 +/- 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/2 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be on the order of 500-650 cm(-1) (+JS(1) . S-2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 +/- 10 and -405 +/- 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced Fe-57-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/2 ground state. Parameters for the ferric site are Delta E(Q) = 1.2 +/- 0.2 mm/s and delta = 0.28 +/- 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, Delta E(Q) = 3.3 +/- 0.1 mm/s and delta = 0.67 +/- 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S](+) cluster-containing proteins are discussed.

Structural information obtained from the analysis of nickel K-edge X-ray absorption spectroscopic data of [NiFe]hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alcaligenes eutrophus H16 (NAD(+)-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme (Volbeda, A.; Charon, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580-587) is compared with the structural features obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutrophus hydrogenase are quite similar. The nickel K-edge energies shift 0.9-1.5 eV to lower energy upon reduction from oxidized (forms A and B) to fully reduced forms. This value is comparable with no more than a one-electron metal-centered oxidation state change. With the exception of T. roseopersicina hydrogenase, most of the edge energy shift (-0.8 eV) occurs upon reduction of the oxidized enzymes to the EPR-silent intermediate redox level (SI). Analysis of the XANES features assigned to 1s-->3d electronic transitions indicates that the shift in energy that occurs for reduction of the enzymes to the SI level may be attributed at least in part to an increase in the coordination number from five to six. The smallest edge energy shift is observed for the T. roseopersicina enzyme, where the XANES data indicate that the nickel center is always six-coordinate. With the exception of the oxidized sample of A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at similar to 2.2 Angstrom. The enzyme obtained from T. roseopersicina also shows evidence for the presence of O,N-donor ligands. The data from A. eutrophus hydrogenase are unique in that they indicate that a significant structural change occurs upon reduction of the enzyme. EXAFS data obtained from the oxidized (as isolated) A. eutrophus enzyme indicate that the EXAFS is dominated by scattering from 3-4 N,O-donor atoms at 2.06(2) Angstrom, with contributions from 2-3 S-donor ligands at 2.35(2) Angstrom. This changes upon reduction to a more typical nickel site composed of similar to 4 S-donor ligands at a Ni-S distance of 2.19(2) Angstrom. Evidence for the presence of atoms in the 2.4-2.9 Angstrom distance range is found in most samples, particularly the reduced enzymes (SI, form C, and R). The analysis of these data is complicated by the fact that it is difficult to distinguish between S and Fe scattering atoms at this distance, and by the potential presence of both S and another metal atom at similar distances. The results of EXAFS analysis are shown to be in general agreement with the published crystal structure of the D. gigas enzyme.

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.

A 7Fe ferredoxin, isolated from the marine denitrifier Pseudomonas nautica strain 617, was characterized. The NH2-terminal sequence analysis, performed until residue number 56, shows a high similarity with the 7Fe ferredoxins isolated from Azotobacter vinelandii, Pseudomonas putida, and Pseudomonas stutzeri. EPR and NMR spectroscopies identify the presence of both [3Fe-4S] and [4Fe-4S] clusters, with cysteinyl coordination. The electrochemical studies on [Fe-S] clusters show that a fast diffusion-dominated electron transfer, promoted by Mg(II), takes place between the ferredoxin and the glassy carbon electrode. Square wave voltammetry studies gave access to the electrosynthesis of a 4Fe center formed within the [3Fe-4S] core. The [3Fe-4S] cluster exhibited two reduction potentials at -175 and -680 +/- 10 mV and the [4Fe-4S] cluster was characterized by an unusually low reduction potential of -715 +/- 10 mV, at pH 7.6

Enoate reductase (EC 1.3.1.31) is a protein isolated from Clostridium tyrobutyricum that contains iron, labile sulfide, FAD, and FMN. The enzyme reduces the alpha,beta carbon-carbon double bond of nonactivated 2-enoates and in a reversible way that of 2-enals at the expense of NADH or reduced methyl viologen. UV-visible and EPR potentiometric titrations detect a semiquinone species in redox intermediate states characterized by an isotropic EPR signal at g = 2.0 without contribution at 580 nm. EPR redox titration shows two widely spread mid-point redox potentials (-190 and -350 mV at pH 7. 0), and a nearly stoichiometric amount of this species is detected. The data suggest the semiquinone radical has an anionic nature. In the reduced form, the [Fe-S] moiety is characterized by a single rhombic EPR spectrum, observed in a wide range of temperatures (4. 2-60 K) with g values at 2.013, 1.943, and 1.860 (-180 mV at pH 7.0). The gmax value is low when compared with what has been reported for other iron-sulfur clusters. Mossbauer studies reveal the presence of a [4Fe-4S]+2/+1 center. One of the subcomponents of the spectrum shows an unusually large value of quadrupole splitting (ferrous character) in both the oxidized and reduced states. Substrate binding to the reduced enzyme induces subtle changes in the spectroscopic Mossbauer parameters. The Mossbauer data together with known kinetic information suggest the involvement of this iron-sulfur center in the enzyme mechanism.

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed.

The three-dimensional X-ray structure of cytochrome c3 from a sulfate reducing bacterium, Desulfovibrio desulfuricans ATCC 27774 (107 residues, 4 heme groups), has been determined by the method of molecular replacement [Frazao et al. (1994) Acta Crystallogr. D50, 233-236] and refined at 1.75 A to an R-factor of 17.8%. When compared with the homologous proteins isolated from Desulfovibrio gigas, Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, and Desulfomicrobium baculatus, the general outlines of the structure are essentialy kept [heme-heme distances, heme-heme angles, His-His (axial heme ligands) dihedral angles, and the geometry of the conserved aromatic residues]. The three-dimensional structure of D. desulfuricans ATCC 27774 cytochrome c3Dd was modeled on the basis of the crystal structures available and amino acid sequence comparisons within this homologous family of multiheme cytochromes [Palma et al. (1994) Biochemistry 33, 6394-6407]. This model is compared with the refined crystal structure now reported, in order to discuss the validity of structure prediction methods and critically evaluate the steps used to predict protein structures by homology modeling. The four heme midpoint redox potentials were determined by using deconvoluted electron paramagnetic resonance (EPR) redox titrations. Structural criteria (electrostatic potentials, heme ligand orientation, EPR g values, heme exposure, data from protein-protein interaction studies) are invoked to assign the redox potentials corresponding to each specific heme in the three-dimensional structure.

All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mossbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mossbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mossbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet. Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.