Characterization of the nickel‐iron periplasmic hydrogenase from Desulfovibrio fructosovorans

Abstract

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 ...

Further characterization of the [Fe]‐hydrogenase from Desulfovibrio desulfuricans ATCC 7757

“…Anaerobic manipulations and conditions for sample preparation were as described previously [23]. 1 U hydrogenase activity in the Hzuptake assay is the amount of enzyme which catalyses the consumption of 1 pmol H2/min [24]. The hydrogen-evolution activity of the purified enzyme was measured at pH 8 and 30°C by the manometric method [25].…”

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

“…When purified aerobically, the so-called "standard" heterodimeric [NiFe] enzymes from the Desulfovibrio genus are inactive and can be activated by reduction. Using EPR or FTIR, the inactive sample appears as a mixture of two forms called NiA and NiB (1)(2)(3). The proposed catalytic cycle of the enzymes involves three intermediates (NiSI, NiC, and NiR) that have only been detected under non-catalytic conditions (2,4).…”

Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

“…In this species, three hydrogenases have been already characterized: a periplasmic [NiFe] hydrogenase which represents about 1% of the total proteins (8,20), a cytoplasmic NADPreducing hydrogenase (13), and a periplasmic [Fe] hydrogenase (4). In order to elucidate the relative importance of these various hydrogenases in the energy-generating metabolism of D. fructosovorans, deletions were first made by marker exchange mutagenesis of the genes encoding the [NiFe] hydrogenase (19) and the NADP-reducing hydrogenase (12).…”