Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H 2 oxidation-to-H 2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H 2 oxidation activity, while having less effect on evolution. The K64,65C3S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H 2 evolution and H 2 oxidation activities with whole cells and membranes of the K294C3S and K297C3S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C3S or K297C3S hydrogenase were uninhibited by O 2 in H 2 oxidation and uninhibited by H 2 in H 2 evolution. Wild-type membranes and membranes with K64,65C3S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O 2 or H 2 .Hydrogenase catalyzes the reversible activation of two electrons from electron carriers and two protons to form molecular hydrogen (1, 2, 7, 11). Azotobacter vinelandii is a free-living, nitrogen-fixing, aerobic, heterotrophic bacterium whose hydrogenase functions in energy conservation by oxidizing H 2 that is evolved by nitrogenase in the course of nitrogen fixation (6,14). A. vinelandii hydrogenase is a member of the [NiFe] hydrogenases and contains approximately one Ni and several Fe atoms per dimer of 67-and 31-kDa polypeptides (6). Ni is thought to be at the active site in [NiFe] hydrogenases (4, 11). The primary evidence for this is electron paramagnetic resonance evidence that hydrogen and CO, a competitive inhibitor against H 2 , both bind the Ni (20, 21). The cysteine ligands to Ni and to a second metal atom close to the Ni (probably Fe) are all on the large polypeptide subunit (5,8,22,23). At least one ligand to Ni, a selenocysteine in some hydrogenases and presumably the homologous cysteine in other hydrogenases, is contributed by the large polypeptide subunit (5,8,23). Other evidence that the active site is on the large subunit is the fact that radioactive acetylene, a competitive inhibitor ...