ESR properties of membrane-bound hydrogenases from aerobic hydrogen bacteria

Abstract

Analysis of the membrane-bound hydrogenase of Alcaligenes eutrophus, strain H16, indicated a content of 7–9 Fe, 7–9 labile sulphide and 0.6–0.7 nickel atoms per molecule of 98 kDa. The protein, as prepared, gave a complex electron-spin resonance (ESR) spectrum in the oxidized state at low temperatures (below 30 K), with average g value greater than 2.0. This was interpreted as being due to the interaction of [3Fe-χS] or [4Fe-4S] 3+ cluster and another paramagnetic centre. At higher temperatures a rhombic spectrum, attributred to Ni(III), was observed. On treatment with mercaptoethanol, or reduction to a redox potential below +160 mV, the low-temperature spectrum changed into a narrow signal at g = 2.02, with increased amplitude. The g = 2.02 signal was reduced reversibly with a midpoint potential of 40 ± 30 mV. The complex spectrum could be restored if the enzyme was rapidly reduced and reoxidized, but not under the conditions of the redox titration, when a broader signal appeared instead. The reductive titration was accompanied by a 3–7-fold increase in enzymic activity. On further reduction of A. eutrophus H16 hydrogenase a complex spectrum with average g value less than 2.0 was observed. This indicate the presence of a [4Fe-45] 1+ cluster, interacting with either another such cluster or another paramagnetic centre. This cluster or clusters were estimated to have a midpoint potential of −90 ± 30 mV. The ESR spectra of the hydrogenases of A. eutrophus, type strain, and Pseudomonas pseudoflava, in the oxidized and reduced states, were similar to those of the A. eutrophus H16 enzyme, indicating that they have a similar arrangement of redox centres. The characteristic spectra of the reduced e indicate that they represent a new type of membrane-bound hydrogenase.