Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution

Abstract

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 ...

Identification of an Uptake Hydrogenase Required for Hydrogen-Dependent Reduction of Fe(III) and Other Electron Acceptors by Geobacter sulfurreducens

“…This cysteine-to-aspartate substitution may have implications for the catalytic activity of Hya. When the analogous cysteine residue of the small subunit of the heterotrimeric respiratory hydrogenase of Azotobacter vinelandii (HoxGKZ) was mutated to serine, the ability of the hydrogenase to catalyze hydrogen oxidation was nearly eliminated (2% of that of the wild type), whereas its ability to catalyze hydrogen evolution was relatively unaffected (22% of that of the wild type) (32).…”

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

“…Identification and engineering of an oxygen-stable hydrogen-evolving hydrogenase might result in a photosynthesizing microorganism that evolves significant amounts of hydrogen. Interestingly, replacing Azotobacter vinelandii hydrogenase small-subunit cysteines with serines can create insensitivity to inhibition by oxygen and preferentially damage hydrogen oxidation over hydrogen evolution (132). Moreover, overproducing mutants might be obtained by providing genes encoding a selected hydrogenase on an expression vector.…”

Hydrogen Evolution by Direct Electron Transfer from Photosystem I to Hydrogenases

“…The native hydrogenase I of C. pasteurianum would be unsuitable from a practical standpoint in any photosynthetic hydrogen production scheme such as this, because it is inhibited and irreversibly inactivated by O 2 . But it might be possible to engineer this hydrogenase for oxygen resistance, as the Azotobacter vinelandii hydrogenase was (25), or a naturally oxygen-resistant hydrogenase such as Rhodococcus sp. MR11 hydrogenase might be used.…”