Purification and characterization of a highly thermostable, oxygen-resistant, respiratory [NiFe]-hydrogenase from a marine, aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus

Abstract The membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus ( Hm MBH) was purified homogeneously under anaerobic conditions. Its molecular weight was estimated as 110 kDa, consisting of a heterodimeric structure of 66 kDa and 37 kDa subunits. The purified enzyme exhibited high activity in a wide temperature range: 185 U/mg at 30 °C and 615 U/mg at 85 °C (the optimum temperature). The K m and k cat / K m values for H 2 were, respectively, 12 μM and 8.58 × 10 7  M −1  s −1 . The optimum reaction pH was 7.8, but its stability was particularly high at pH 4.0–7.0. Results show that Hm MBH was remarkably thermostable and oxygen-resistant: its half-life was 75 h at 80 °C under H 2 , and more than 72 h at 4 °C under air. The air-oxidized Hm MBH for 72 h showed only weak EPR signals of Ni–B, suggesting a structural feature in which the active center is not easily oxidized.