Purification and properties of the membrane - bound hydrogenase from Desu ( fovibrio desufwricans

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70,umol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The 400and 6280 were 28 500 and 109 000MW-1 cm-' respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3SI3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to 02 over long periods and to high temperatures. It catalyses both H2-evolution and H2uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2,UM). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2evolution assay. The pH optimum for H27evolution was 6.5.