The same polypeptide chain (58 amino acids, 6 cysteines) is used to build up two ferredoxins in Desulfovibrio gigas a sulfate reducing organism. Ferredoxin II (FdII) contains a single [Fe3S4] core and ferredoxin I (FdI) mainly a [Fe4S4] core. The [Fe3S4] core can readily be interconverted into a [Fe4S4] complex (J.J.G. Moura, I. Moura, T.A. Kent, J.D. Lipscomb, B.H. Huynh, J. LeGall, A.V. Xavier, and E. Munck, J. Biol. Chem. 257, 6259 (1982)). This interconversion process suggested that the [Fe3S4] core could be used as a synthetic precursor for the formation of heterometal clusters. Co, Zn, Cd, and Ni derivatives were produced (I. Moura, J.J.G. Moura, E. Munck, V. Papaephthymiou, and J. LeGall, J. Am. Chem. Soc. 108, 349 (1986), K. Sureurs, E. Munck, I. Moura, J.J.G. Moura, and J. LeGall, J. Am. Chem. Soc. 109, 3805 (1986), and A.L. Macedo, I. Moura, J.J.G. Moura, K. Surerus, and E. Munck, unpublished results). The redox properties of a series of heterometal clusters (MFe3S4] are assessed using direct electrochemistry (square wave voltammetry--SWV) promoted by Mg(II) at a glassy carbon electrode (derivatives: Cd (-495 mV), Fe (-420 mV), Ni (-360 mV), and Co (-245 mV) vs normal hydrogen electrode (NHE)). In parallel, the electrochemical behavior (cyclic voltammetry--CV, differential pulse voltammetry--DPV and SWV) of FdI and FdII were investigated as well as the cluster interconversion process. In addition to the +1/0 (3Fe cluster) and +2/+1 (4Fe cluster) redox transitions, a very negative redox step, at -690 mV, was detected for the 3Fe core, reminiscent of a postulated further 2e- reduction step, as proposed for D. africanus ferredoxin III by F.A. Armstrong, S.J. George, R. Cammack, E.C. Hatchikian, and A.J. Thomson, Biochem. J. 264, 265 (1989). The electrochemical redox potential values are compared with those determined by independent methods (namely by electron paramagnetic resonance (EPR) and visible spectroscopy).

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R.S. & Shain, I. (1964) Anal. Chem. 36, 706-723], using the Gauss-Legendre method for calculation of the integral equations. The experimental data obtained for this multi-redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid-point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described.

Formate dehydrogenases (FDHs) are enzymes that catalyze the formate oxidation to carbon dioxide and that contain either Mo or W in a mononuclear form in the active site. In the present work, the influence of Mo and W salts on the production of FDH by Desulfovibrio alaskensis NCIMB 13491 was studied. Two different FDHs, one containing W (W-FDH) and a second incorporating either Mo or W (Mo/W-FDH), were purified. Both enzymes were isolated from cells grown in a medium supplemented with 1 muM molybdate, whereas only the W-FDH was purified from cells cultured in medium supplemented with 10 muM tungstate. We demonstrated that the genes encoding the Mo/W-FDH are strongly downregulated by W and slightly upregulated by Mo. Metal effects on the expression level of the genes encoding the W-FDH were less significant. Furthermore, the expression levels of the genes encoding proteins involved in molybdate and tungstate transport are downregulated under the experimental conditions evaluated in this work. The molecular and biochemical properties of these enzymes and the selective incorporation of either Mo or W are discussed.

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferrodoxin protein from Desulphovibrio gigas, FdI, FdI', and FdII was carried out. FdI and FdI' are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted responances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the "three state hypothesis" terminology it is shown that FdIox is predominantly in the C2- state and changes upon reduction into the C3- state, while FdIIox is in the C- state and changes into the C2- state. FdI' does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.

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.

Methanosarcina barkeri ferredoxin was purified and characterized by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. The purification procedure included chromatographic steps on DEAE-cellulose and gel filtration. The isolated protein is unstable under aerobic conditions. The ferredoxin exhibits charge transfer bands at 283 nm and 405 nm with an absorption ratio A405/A283 = 0.73. Its molecular weight has been estimated to be 20000-22000 by gel filtration chromatography. The native ferredoxin exhibits an intense EPR signal at g = 2.02 and only a very weak g = 1.94 signal develops upon reduction with dithionite. The Mossbauer spectra of the reduced protein are characteristic of a [3Fe-3S] center. The combined EPR and Mossbauer studies show that M. barkeri ferredoxin contains only [3Fe-3S] clusters, similar to Azotobacter vinelandii Fd[Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H. & Munck, M. (1980) J. Biol. Chem. 255, 1793-1796], Desulfovibrio gigas FdII [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V. & Munck, E. (1980) J. Biol. Chem. 255, 3242-3244] and mitochondrial beef heart aconitase [Kent, T.A., Dreyer, J.-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H. & Munck, E. (1982) Proc. Natl Acad. Sci. USA, 79, 1096-1100].

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

A flavodoxin and a rubredoxin have been isolated from the sulfate-reducing bacterium Desulphovibrio salexigens (strain British Guiana, NICB 8403). Their amino acid composition and spectral characteristics did not differ markedly from the homologous proteins presented in other Desulphovibrio spp. Flavodoxin was shown to be active in the electron transport of the sulfite reductase system.

Pseudomonas perfectomarinus (ATCC 14405) dihaem cytochrome c552 was studied by 300-MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation-reduction equilibrium was studied in detail. Due to the large difference in mid-point redox potential between the two haems (+174 mV, for haem II and -180 mV for haem I) an intermediate oxidation state could be obtained containing reduced haem I and oxidized haem II. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems interact. The rate of electron exchange is slow on the NMR time scale. The redox equilibria are discussed for four possible redox species in solution.

Two c-type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate-reducer nitrogen-fixing organism, Desulfovibrio desulfuricans strain Berre-Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low-potential bis-histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate-reducible and its EPR and NMR data are characteristic of a cytochrome with methionine-histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate-reducing bacteria.

1H nuclear magnetic resonance (NMR) spectroscopy has been used to examine cytochrome c551.5 (c7) from the sulfur reducer, Desulfuromonas acetoxidans. This protein contains three hemes. Two stable oxidation states (the fully oxidized and the fully reduced) as well as intermediate oxidation states were studied. The axial ligands of the iron were found to be neutral histidines. The redox properties of cytochrome c7 were examined and good quantitative agreement found between the NMR results and previously reported redox potential measurements. The properties of cytochrome c7 are discussed together with those of the homologous tetraheme cytochromes c3 isolate from sulfate-reducing bacteria.

An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmx = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results.