X-ray absorption spectroscopy of nickel in the hydrogenase from Desulfovibrio gigas

Scott, Robert A.; Wallin, Sten A.; Czechowski, Melvin H.; Vartanian, Daniel V. Der; LeGall, Jean; Peck, Harry D.; Moura, Isabel

doi:10.1021/ja00334a078

Cited by 95 publications

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“…D . fructosovorans hydrogenase has a molecular mass of 89 kDa and consists of two subunits of different sizes (60 kDa and 28.5 In hydrogenases, cysteine residues are particularly relevant, since they are involved in the binding of the iron-sulfur clusters and the nickel centre to the protein [35, 49,[59][60][61]. The distribution of the cysteine residues of D. fructosovorans hydrogenase between the large and the small subunits suggests that both subunits could contribute sulfur ligands for binding the four metal centres.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the nickel‐iron periplasmic hydrogenase from Desulfovibrio fructosovorans

Hatchikian¹,

Traoré²,

Fernández³

et al. 1990

European Journal of Biochemistry

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The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-' cm-', respectively. D. fructosovorans hydrogenase contains 11 f 1 iron atoms, 0.9 f 0.15 nickel atom and 12 f 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D . gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.Sulfate-reducing bacteria exhibit a strictly anaerobic mode of growth based on reduction of sulfate as the terminal electron acceptor [l]. Hydrogen metabolism plays a central role in energy-generating mechanisms of these microorganisms [2 -61. Hydrogen cycling with vectorial electron transfer has been proposed as a general mechanism for energy coupling for Desulfovibrio species during growth on organic substrates in the presence of sulfate [5, 61. Hydrogenase (EC 1.18.99.1), which catalyzes the reversible oxidation of molecular hydrogen ( H 2 e 2 H t + 2e-), is the key enzyme of hydrogen metabolism.Three Most of hydrogenases isolated from these bacteria have been found to be confined to the periplasmic space [9,16 -191 ; however, membrane-bound and cytoplasm-located enzymes have also been described [12,20,21]. The existence of multiple forms of hydrogenases within a single species of Desulfovibrio [9, 12, 20,221 is a matter of particular relevance in view of the current discussion about the bioenergetics of these microorganisms and their role in interspecies hydrogen transfer [2, 51. Catalytic and activation schemes have already been proposed, showing ...

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Section: Discussionmentioning

confidence: 99%

Characterization of the nickel‐iron periplasmic hydrogenase from Desulfovibrio fructosovorans

Hatchikian¹,

Traoré²,

Fernández³

et al. 1990

European Journal of Biochemistry

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“…Previous EXAFS studies have demonstrated sulphur coordination to the metal [25,26]. It was implied [26] that lighter atom coordination could be masked by the predominance of sulphur scattering in the data, making complete structural assignment difficult.…”

Section: Discussionmentioning

confidence: 99%

A pulsed EPR study of redox‐dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase

et al. 1988

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The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni‐A) and H2‐reduced active (Ni‐C) states, both in H2O and 2H2O solution. Fourier transforms of the 3‐pulse ESEEM, taken at 8.7 GHz, for Ni‐A and Ni‐C in H2O contained similar peaks with narrow linewidths at frequencies of 0.4, 1.2 and 1.6 MHz, and a broader peak centred at 4.5 MHz. At 11.6 GHz, the low frequency components showed small field‐dependent shifts, while the high frequency component was shifted to 5.1 MHz. These results are consistent with the presence of 14N, possibly from imidazole, coupled to the nickel centre. In 2H2O, Ni‐A was shown to be inaccessible for exchange with solvent deuterons. In contrast, Ni‐C was accessible to solvent exchange, with a deuterium population being in close proximity to the metal ion. Thus, the nickel environment of the active protein is different from that in the oxidized or unready state. On illumination of Ni‐C, although EPR changes are seen, 14N coupling remains, and for the 2H2O sample, deuterium coupling is also retained.

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“…The sample for x-ray absorption spectroscopy (XAS) was placed in a Lucite/Mylar cell, -80 gl in volume, and stored in liquid nitrogen until the time of data collection. The Desulfovibrio gigas [NiFeihydrogenase sample preparation has been described (22 tTo whom reprint requests should be addressed.…”

Section: Methodsmentioning

confidence: 99%

Evidence for selenocysteine coordination to the active site nickel in the [NiFeSe]hydrogenases from Desulfovibrio baculatus.

Eidsness

Scott

Prickril

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

117

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Ni and Se x-ray absorption spectroscopic studies of the [NiFeSeihydrogenases from Desulfovibrio baculatus are described. The Ni site geometry is pseudo-octahedral with a coordinating ligand composition of 3-4 (N,O) at 2.06 A, 1-2 (S,CI) at 2.17 A, and 1 Se at 2.44 A. The Se coordination environment consists of 1 C at 2.0 A and a heavy scatterer M (M = Ni or Fe) at -2.4 A. These results are interpreted in terms of a selenocysteine residue coordinated to the Ni site. The possible role of the Ni-Se site in the catalytic activation of H2 is discussed.Hydrogenases are vital to the anaerobic metabolism of sulfate-reducing bacteria and many other types of bacteria. Hydrogenases catalyze the bidirectional activation of molecular hydrogen H2 = 2H+ + 2e-and their activities are routinely determined by H2 production, H2 utilization, or the 2H2-H+ exchange assays (1). In the past, it was generally accepted that a single hydrogenase carried out these simple redox reactions (1), but in recent years the unveiling of the structural diversity of hydrogenases has promoted the idea that different hydrogenases may reflect differences in cellular localization and metabolic function (2). This specificity suggests that structural differences are the basis for tailoring the hydrogenase reaction to meet metabolic demands. The elucidation of the structural details of the active sites of hydrogenases is the first step toward a molecular understanding of the mechanisms involved in the hydrogenase reaction.In the genus Desulfovibrio, the metabolism of hydrogen involves at least three types of hydrogenases that may be distinguished by their heavy element composition, immunological reactivities, and gene structures (3-6). The [Fe]hydrogenases contain only iron-sulfur clusters (7-9), the [NiFe] As part of our effort to elucidate the structures of the nickel sites in the Ni-containing hydrogenases, and to define a structural basis for the functional differences between the Seand the non-Se-containing hydrogenases (19, 20), we report here the results of Ni and Se x-ray absorption spectroscopic measurements on the [NiFeSe]hydrogenases ofD. baculatus. MATERIALS AND METHODS Sample Preparation. The D. baculatus [NiFeSe]hydrogenasesample was prepared according to the procedure described in ref. 19. To obtain a sample -2 mM Ni and Se, the periplasmic, cytoplasmic, and membrane-bound fractions were combined. Total iron was determined by the 2,4,6-tripyridyl-1,3,5-triazine method (21), and metals were quantified by plasma emission spectroscopy using a Mark II Jarrell-Ash model 965 AtomComp (Fisher). Nickel was also determined by atomic absorption spectroscopy. The sample for x-ray absorption spectroscopy

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X-ray absorption spectroscopy of nickel in the hydrogenase from Desulfovibrio gigas

Cited by 95 publications

References 0 publications

Characterization of the nickel‐iron periplasmic hydrogenase from Desulfovibrio fructosovorans

Characterization of the nickel‐iron periplasmic hydrogenase from Desulfovibrio fructosovorans

A pulsed EPR study of redox‐dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase

Evidence for selenocysteine coordination to the active site nickel in the [NiFeSe]hydrogenases from Desulfovibrio baculatus.

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