PUB - Publikationen an der Universität Bielefeld

For highly active preparations of the membrane-bound, respiratory chain-linked hydrogenase from Alcaligenes eutrophus strains H16 and CH34, 0.7 moles of nickel and approximately 10 moles of iron per mole of enzyme were determined. The hydrogenase isolated from A. eutrophus CH34, displayed the highest specific activity (540 U/mg protein) and the most intense Ni signals and was therefore used to study the redox properties of the metal centres present in this type of hydrogenase. At 80 K two well-resolved Ni spectra were detected in the course of a redox titration (at pH 7.0): one displayed a Ni-B type signal at g = 2.30, 2.17 and 2.01 representing the Ni(III) centre in the ''ready state'' of the enzyme (''active'' protein conformation, oxidized catalytic centre), the other one consisted of a Ni-C type signal at g = 2.20, 2.16 and 2.01 apparently representing monovalent nickel in the ''active state'' of the enzyme (''active'' protein conformation, reduced catalytic centre) and a minor proportion of a second Ni-C signal (g = 2.31, 2.12, 2.05) induced by the influence of normal daylight. The midpoint potentials determined are -30 mV for Ni(III), -260 mV (appearance of the Ni-C signal) and -340 mV (disappearance of the Ni-C signal) for apparent Ni(I). The midpoint potentials calculated for the FeS clusters are: + 100 mV ([3Fe-4S]1+/0), + 50 m V ([4Fe-4S]2+/1+ cluster I) and -80 mV ([4Fe-4S]2+/1+ cluster II).