Comparison of the membrane-bound hdyrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Abstract

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus II16 is an integral membrane protein an can only be solubilized by detergent treatment, the membrane-bound hdyrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus II16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl 2. The hydrogenase of A. eutrophus type was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits ( M r = 62000, 31000 ) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar M m values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrohoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H 2 evolution from reduced methyl viologen.