Content and localization of FMN, Fe‐S clusters and nickel in the NAD‐linked hydrogenase of Nocardia opaca 1b

Abstract

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD‐linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 ± 0.4 labile sulphur, 13.6 ± 1.1 iron and 3.8 ± 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g= 2.01. The midpoint redox potential of the Fe‐S cluster giving rise to this signal was + 25 m V. In the reduced state, hydrogenase gave characteristic low‐temperature (10 – 20 K) and high‐temperature (>40 K) ESR spectra which were interpreted as due to [4Fe–4S] and [2Fe–2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be ‐420 mV and ‐285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses M_r of 64000 and 31000. contained 9.9 ± 0.4 S²⁻ and 9.3 ± 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with M_r of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400‐nm region and a characteristic ESR signal at g= 2.01. The reduced form of the dimer was ESR‐silent. The small dimer alone was diaphorase‐inactive and did not reduce NAD with H₂, but it displayed high H₂‐uptake activities with viologen dyes, methylene blue and FMN, and H₂‐evolving activity with reduced methyl viologen. Hydrogen‐dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the, ESR spectrum in the oxidized state.