The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus

Abstract

The soluble hydrogenase (hydrogen:NAD + oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 μmol H 2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H 2. Chelating agents, CO and O 2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50–77 K) signal at g = 2.04, 1.95 and a more complex low temperature (<30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80–85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be −445 mV and −325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [4Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. of hydrogenase with CO caused no change in EPR spectra.