The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g x 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(II1). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and x 2.0 (component I) and at 2.33, 2.24, and x 2.0 (component 11). In the reduced state, after exposure to an Hz atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers : center I (2.03, 1.89 and 1.86) detectable below 10 K, and center I1 (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 89421. During the course of a redox titration at pH 7.6 using Hz gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fastrelaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymatic activity. The catalytic properties were also followed by the proton-deuterium exchange reaction. The isolated hydrogenases produced Hz/HD ratios higher than those observed for nonselenium-containing hydrogenases.The enzyme responsible for the biological activation of H2, termed hydrogenase [l, 21, has a central role in many relevant anaerobic processes where molecular hydrogen is oxidized or evolved. Also, molecular hydrogen, via the hydrogenase system, is a link between different bacterial consortia w...

This paper presents a Q-band ENDOR study of the nickel site of the as-isolated (Ni-A), H2-reduced (Ni-C), and reoxidized (Ni-A/Ni-B) states of Desulfovibrio gigas hydrogenase. Through proton and deuteron ENDOR measurements we detect and characterize the possible products of heterolytic cleavage of H2, namely two distinct types of exchangeable protons, bound to the Ni-C site. One proton, H(l), has a hyperfine coupling, AH( 1) = 16.8 MHz and appears to interact directly with Ni-C. The other proton, H(2), has AH(2) =» 4.4 MHz and could be associated with H20 or OH" bound to nickel. We discuss possible binding modes for H(l) and H(2). One type of exchangeable deuteron(s), D(2), associated with the Ni-C center remains associated with the Ni-B center after oxidation of the Ni-C. In addition we confirm that the Ni-A site is inaccessible to solvent protons.

A novel iron-containing blue protein, named neelaredoxin, was isolated from the sulfate-reducing bacterium Desulfovibrio gigas. It is a monomeric protein with a molecular mass of 15 kDa containing two iron atoms/molecule. The N-terminal sequence of neelaredoxin has similarity to the second domain of desulfofen-odoxin, a protein purified from Desulfovibrio vulgaris Hildenborough. This finding supports the hypothesis that the gene coding for desulfoferrodoxin (rbo) might have arisen from a gene fusion [Brumlik, M. J., Leroy, G., Bruschi, M. & Voordouw, G. (1990) J. Bacteriol. 172, 7289-72921. The visible spectrum exhibits a single band at 666 nm, responsible for the blue color of the protein, which is completely bleached upon reduction with sodium ascorbate. In the oxidized state the EPR spectrum is complex, exhibiting well-resolved features at g = 7.6, 7.0, 5.9, and 5.8 which are assigned to two high-spin (5' = 5/2) mononuclear-iron (111) centers with different rhombic distortions (EID = 0.05 and = 0.08). The two iron atoms contribute identically to the visible spectrum as judged from visible redox titrations, from which a reduction potential of +190 mV was determined for both iron sites at pH 7.5. At high pH the visible and the EPR spectra become pH-dependent with a pK, above 9 : the 666-nm band shifts to 590 nm and the EPR signals are converted into a signal with g, , , = 4.7. Neelaredoxin is readily reduced both by H2/hydrogenase/ cytochrome c3 and by NADWNADH -rubredoxin oxidoreductase.Sulfate-reducing bacteria are rich in electron-carrier proteins. These proteins can be classified into three groups : flavoproteins, hemoproteins, and non-heme iron proteins. Detailed discussions concerning these proteins can be found in recent reviews [I, 21. Within the group of non-heme-iron proteins, besides the iron-sulfur-containing ferredoxins, several redox proteins with no labile sulfur have been discovered containing mononuclear or dinuclear iron centers. These include rubredoxin, desulforedoxin, desulfoferrodoxin, rubrerythrin and nigerythrin.Rubredoxin is the smallest protein (Mr = 6000) found in sulfate-reducing bacteria including Desulfovibrio gigas [3], D. vulgaris Hildenborough [4], D. desulfuricans [5] and D.salexigens [6]. The structure of the protein has been well studied [7]. This protein contains one iron atodmolecule which links to four cysteinyl residues in the polypeptide chain. Its high reduction potential (-50 to 0 mV) made it difficult to find an appropriate place in any electron transfer chain. Recently, D. gigas rubredoxin was shown to function in an aerobic electron transfer chain which allows ATP formation from polyglucose [S, 91. Desulforedoxin is another [Fel-only-

This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.

The molecular structure of cytochrome c3 isolated from Desulfovibrio desulfuricans has been solved on the basis of its crystallographic determination at 2.5 A resolution and of an essentially complete sequence. The molecule consists of a single polypeptide chain wrapped around a very compact core of four non-parallel haems which present a relatively high degree of exposure to the solvent. Alignment of the amino acid sequences of cytochrome c3 from several species suggests that the structure reported here is characteristic of the cytochrome c3 group.

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