Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1b

Abstract

The NAD‐reducing hydrogenase of Nocardia opaca 1b was found to be a soluble, cytoplasmic enzyme. N. opaca 1b does not contain an additional membrane‐bound hydrogenase. The soluble enzyme was purified to homogeneity with a yield of 19% and a final specific activity of 45 μmol H₂ oxidized min⁻¹ mg protein⁻¹. NAD reduction with H₂ was completely dependent on the presence of divalent metal ions (Ni²⁺, Co²⁺, Mg²⁺, Mn²⁺) or of high salt concentrations (0.5–1.5 M). The most specific effect was caused by NiCl₂, whose optimal concentration turned out to be 1 mM. The stimulation of activity by salts was the greater the less chaotrophic the anion. Maximal activity was achieved in 0.5 M potassium phosphate. Hydrogenase was also activated by protons. The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiCl₂ was 7.8–8.0. In the absence of Ni²⁺, hydrogenase was only active at pH values below 7.0. The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1.5‐fold stimulation of the reactions by 0.1–10 μM NiCl₂ was observed. With the most effective electron acceptor, benzyl viologen, a 50‐fold higher specific activity was determined than with NAD. The total molecular weight of hydrogenase has been estimated to be 200000 (gel filtration) and 178000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively. The enzyme is a tetramer consisting of non‐identical subunits with molecular weights of 64000, 56000, 31000 and 27000. It was demonstrated by electrophoretic analyses that in the absence of NiCl₂ and at alkaline pH values the native hydrogenase dissociates into two subunit dimers. The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64000 and 31000. The second dimer was light yellow, inactive with NAD but still active with methyl viologen. It was composed of subunits with molecular weights of 56000 and 27000. Immunological comparison of the hydrogenase of N. opaca 1b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD‐linked hydrogenases are partially identical proteins.