The complete amino acid sequence of the unusual diheme split-Soret cytochrome c from the sulphate-reducing Desulfovibrio desulfuricans strain ATCC 27774 has been determined using classical chemical sequencing techniques and mass spectrometry. The 247-residue sequence shows almost no similarity with any other known diheme cytochrome c, but the heme-binding site of the protein is similar to that of the cytochromes c3 from the sulphate reducers. The cytochrome-c-like domain of the protein covers only the C-terminal part of the molecule, and there is evidence for at least one more domain containing four cysteine residues, which might bind another cofactor, possibly a non-heme iron-containing cluster. This domain is similar to a sequence fragment of the genome of Archaeoglobus fulgidus, which confirms the high conservation of the genes involved in sulfate reduction.

Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A. Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55-60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) = 32 microM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3-C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD).

The formate dehydrogenase (FDH) isolated from cells of Methylobacterium sp. RXM grown on molybdenum-containing mineral medium using methanol as carbon source, was partially purified (at least 90% pure as revealed by SDS-PAGE). The enzyme is unstable under oxygen and all the purification steps were conducted under strict anaerobic conditions. The molecular mass is 75 kDa (gel exclusion 300 kDa). The enzyme was characterized in terms of the kinetic parameters towards different substrates and electron acceptors, pH and temperature dependence and the effect of a wide range of compounds in the enzymatic activity. The EPR spectra of the dithionite reduced sample show, at low temperature (below 20 K), two rhombic EPR signals due to two distinct [Fe-S] centres (centre I at g-values 2.023, 1.951 and 1.933, and centre II at g-values 2.054 and 1.913). At high temperature (around 100 K) another rhombic EPR signal is optimally observed at g-values 2.002, 1.987 and 1.959 and attributed to the molybdenum site. The EPR signals assigned to the iron-sulfur centres show a strong analogy with the aldehyde oxido-reductase from Desulfovibrio gigas known to contain a Mo-pterin and two [2Fe-2S] centres and whose crystallographic structure was recently resolved.

Rubredoxin and desulforedoxin both contain an Fe(S-Cys)4 center. However, the spectroscopic properties of the center in desulforedoxin differ from rubredoxin. These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated highspin Fe3+ ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin.

An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO2, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150 kDa (three different subunits: 88, 29 and 16 kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S](2+/1+) centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g(max)=2.050, g(med)=1.947, g(min)=1.896) and an [Fe-S] center II (g(max)=2.071, g(med)=1.926, g(min)=1.865). Mossbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S](2+/1+) centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.

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.

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Rubredoxin and desulforedoxin both contain an Fe(S-Cys)(4) center, However the spectroscopic properties of the center in desulforedoxin differ from rubredoxin, These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated high-spin Fe3+ ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin. (C) 1997 Academic Press.

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The dissimilatory nitrite reductase from Desulfovibrio desulfuricans ATCC 27774 catalyzes the reduction of nitrite to ammonia. Previous spectroscopic investigation revealed that it is a hexaheme cytochrome containing one high spin ferric heme and five low spin ferric hemes in the oxidized enzyme. The current study uses the high resolution of Mossbauer spectroscopy to obtain redox properties of the six heme groups. Correlating the Mossbauer findings with the EPR data reveals the pairwise spin-spin coupling among four of the heme groups. The other two hemes are found to be magnetically isolated. Reduction with dithionite and reaction with CO further indicate that only the high spin heme is capable of binding small exogenous ligands. These results confirm our previous finding that Desulfovibrio desulfuricans nitrite reductase contains six heme groups and that the high spin ferric heme is the substrate and inhibitor binding site.

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.

Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions. Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains. Its molecular mass (37768 kDa) was determined by electrospray mass spectrometry. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c(3) type from sulfate-reducing bacteria sequenced at present. The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV. Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983. Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5. Both crystals belong to monoclinic space groups: one is P2(1), with a = 61.00, b = 106.19, c = 82.05 A, beta = 103.61 degrees, and the other is C2 with a = 152.17, b = 98.45, c = 89.24 A, beta = 119.18 degrees. Density measurements of the P2(1) crystals suggest that there are two independent molecules in the asymmetric unit. Self-rotation function calculations indicate, in both crystal forms, the presence of a non-crystallographic axis perpendicular to the crystallographic twofold axis. This result and the calculated values for the volume per unit molecular weight of the C2 crystals suggest the presence of two or four molecules in the asymmetric unit.