Reinvestigation of regulation of biosynthesis and subunit composition of nickel-dependent Hup-hydrogenase of Rhodospirillum 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), NH 4 + 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 Ni 2+ (80–150 μM). Activation of hydrogenase biosynthesis by Ni 2+ was completely inhibited by chloramphenicol. The enzyme, solubilized from intracytoplasmic membranes by Triton X-114 and partially purified by gelfiltration, had a M r of about 100 000. When analysed by SDS-PAGE, the native enzyme yielded three different protein bands with M r values of 40 000, 34 000 and 29 000.