Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans

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Abstract
The membrane-bound hydrogenase from the anaerobic sulfate-reducing bacterium D. desulfuricans (Norway strain) was purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 .mu.mol of H2 evolved/min per mg of protein. The hydrogenase had a relative MW of 58,000 as determined by gel filtration and was estimated to contain 6 Fe atoms and 6 acid-labile S groups per molecule. The absorption spectrum of the enzyme was characteristic of an Fe-S protein. The .epsilon.400 [molar extinction coefficient] and .epsilon.280 were 28,500 and 109,000 M-1 .cntdot. cm-1, respectively. The ESR of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic center, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, CO and high ionic strength. It was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at a low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyzes both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome c3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 .mu.M). Rate enhancement was observed when cytochrome c3 was added to methyl viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.