[NiFeSe]hydrogenases are promising biocatalysts in H-2-based technology due to their high catalytic activity and O-2-stability. Here, we report purification and characterization of a new membrane-associated [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F ([NiFeSe]DvMF). The [NiFeSe]DvMF was composed of two subunits, corresponding to a large subunit of 583 kDa and a small subunit of 29.3 kDa determined by SDS-PAGE. Unlike conventional [NiFeSe] hydrogenases having catalytic bias toward H-2-production, the [NiFeSe]DvMF showed 11-fold higher specific activity of H-2-oxidation (2444 U/mg) than that of H-2-production (217 U/mg). At the optimal reaction temperature of the enzyme (65 degrees C), the specific activity of H2-oxidation could reach up to 21,553 U/mg. Amperometric assays of the [NiFeSe]DvMF clearly indicated that the enzyme had a remarkable O-2-stability. According to the amino acid sequence alignment, the conserved cysteine residue at position 281 in medial cluster of other [NiFeSe]hydrogenases was specifically replaced by a serine residue (Ser281) in the [NiFeSe]DvMF. These results indicate that the [NiFeSe]DvMF can play as a new H-2-oxidizing and O-2-stable biocatalyst, along with providing helpful insights into the structure-function relationship of [NiFeSe] hydrogenases. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.