SUBSTITUTION OF AZOTOBACTER-VINELANDII HYDROGENASE SMALL-SUBUNIT CYSTEINES BY SERINES CAN CREATE INSENSITIVITY TO INHIBITION BY O-2 AND PREFERENTIALLY DAMAGES H-2 OXIDATION OVER H-2 EVOLUTION

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 K64,65C-->S 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 K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity, Membranes with K294C-->S or K297C-->S hydrogenase mere uninhibited by O-2 in H-2 oxidation and uninhibited by H-2 in H-2 evolution, Wild-type membranes and membranes with K64,65C-->S 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.