Purification and properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans

Abstract

The periplasmic hydrogenase of Desulfovibrio desulfuricans was isolated and purified. Cells were washed with Tris–EDTA and the enzyme precipitated from the wash with ammonium sulfate. Absorption chromatography on DEAE and hydroxyapatite yielded the enzyme at better than 95% purity as judged by gel electrophoresis. The hydrogenase catalyzed the production of more than 9000 μmol H2/min mg protein−1 from reduced methyl viologen at 37 °C. It is very stable and resists inactivation by heat (50% activity remained after 5 min in air at 65 °C) and by enzyme inhibitors (except N-ethylmaleimide and potassium ferricyanide). After storage in air at 4 °C for 1 month no activity was lost. The enzyme activity is sensitive to ionic environmental changes. With methyl viologen the optimum pH was 5.5 but with p-xylene polymeric viologen the optimum was about pH 7 but less sharp. The molecular weight was 47 × 103 (± 2 × 103), 52 × 103(± 2 × 103), and 56 × 103 (± 2 × 103) by SDS –gel electrophoresis, gel chromatography, and sedimentation equilibrium, respectively, and the isoelectric point was at pH 6.0. The enzyme consists of one polypeptide chain terminated at the amino end by proline. This enzyme might be useful in the production of hydrogen from water and solar energy.