Reinvestigation of regulation of biosynthesis and subunit composition of nickel-dependent Hup-hydrogenase ofRhodospirillum rubrum

Abstract

Regulation of biosynthesis and structural properties of the membrane-bound ‘hydrogen uptake’ (Hup)-hydrogenase of Rhodospirillum rubrum were studied. Under photoheterotrophic conditions, the highest activities were recorded in media with L-lactate, the lowest activities with malate. At high concentrations (20–40 mM), NH4+ or glutamate suppressed biosynthesis of hydrogenase. In culture media supplemented with 0.5 mM EDTA, normal Hup activities were only obtained in the presence of high concentrations of Ni2+ (80–150 μM). Activation of hydrogenase biosynthesis by Ni2+ was completely inhibited by chloramphenicol. The enzyme, solubilized from intracytoplasmic membranes by Triton X-114 and partially purified by gelfiltration, had a Mr of about 100 000. When analysed by SDS-PAGE, the native enzyme yielded three different protein bands with Mr values of 40 000, 34 000 and 29 000.