Properties of the particulate enzyme F420-reducing hydrogenase isolated from Methanospirillum hungatei

F420-reducing hydrogenase was isolated from spheroplast lysates of Methanospirillum hungatei by sedimentation, followed by sucrose gradient centrifugations and gel filtration. These procedures resulted in a preparation free of methyl reductase and cytoplasmic membranes. The hydrogenase was a brown protein with an absorption spectrum characteristic for a nonheme iron protein. In electron micrographs it was a coin-shaped, multisubunit protein complex of 15.9 nm diameter with a central depression on one surface. On phenyl Sepharose chromatography the hydrogenase exhibited hydrophobic properties. The holoenzyme was about 720 kilodaltons (kDa), composed of 50.7 and 30.7 kDa subunits in a ratio of 1:3. Each enzyme particle contained 6 or 7 Ni2+ atoms. H2-dependent reduction of F420 activity was readily, but transiently, reactivated by anaerobic conditions following exposure of the enzyme to air. Mg2+ or Ca2+ were stimulatory, but added FAD was not required. Antibody raised against the purified hydrogenase of st...