Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.

A c-type monohemic ferricytochrome c552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome c552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome c552 is typical of a low-spin ferric heme.

On photolysis of solutions of azulene and uranyl nitrate in alcohols, a dark, amorphous precipitate is formed. Various analytical techniques show that this is a mixture of a uranium salt and an organic component, suggested to be polyazulene. The effects of various parameters on the yield of the product have been studied and it is found that oxygen facilitates the reaction. Electron spin resonance studies show that the product is paramagnetic, in agreement with the established ease of oxidation of polyazulene, and suggest that it is formed via electron transfer from azulene to excited uranyl ion, followed by successive dimerizations and deprotonations of radical cation intermediates.

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.

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.

A dissimilatory bisulfite reductase has been purified from a thermophilic sulfate-reducing bacterium Desulfovibrio thermophilus (DSM 1276) and studied by EPR and optical spectroscopic techniques. The visible spectrum of the purified bisulfite reductase exhibits absorption maxima at 578.5, 392.5 and 281 nm with a weak band around 700 nm. Photoreduction of the native enzyme causes a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm. When reduced, the enzyme reacts with cyanide, sulfite, sulfide and carbon monoxide to give stable complexes. The EPR spectrum of the native D. thermophilus bisulfite reductase shows the presence of a high-spin ferric signal with g values at 7.26, 4.78 and 1.92. Upon photoreduction the high-spin ferric heme signal disappeared and a typical 'g = 1.94' signal of [4Fe-4S] type cluster appeared. Chemical analyses show that the enzyme contains four sirohemes and eight [4Fe-4S] centers per mol of protein. The molecular mass determined by gel filtration was found to be 175 kDa. On SDS-gel electrophoresis the enzyme presents a main band of 44 to 48 kDa. These results suggest that the bisulfite reductase contains probably one siroheme and two [4Fe-4S] centers per monomer. The dissimilatory bisulfite reductase from D. thermophilus presents some homologous properties with desulfofuscidin, the bisulfite reductase isolated from Thermodesulfobacterium commune (Hatchikian, E.C. and Zeikus, J.G. (1983) J. Bacteriol. 153, 1211-1220).

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.

Three forms of ferredoxin FdI, FdI', and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI' present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI' and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI' and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.