Purification and properties of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum.

Abstract

A coenzyme F430-reducing hydrogenase from Methanobacterium thermoautotrophicum has been purified 25-fold, to approximately 50% homogeneity. Following anaerobic preincubation in high salt under reducing conditions, the purified enzyme exhibits equal catalytic activity (turnover number = 725 s-1) toward the artificial 1-electron acceptor, methyl viologen, and the physiological 2-electron acceptor, 7,8-didemethyl-8-hydroxy-5-deazaflavin (F420). The enzyme had the following Km values (micromolarity) under the described assay conditions: 420 (methyl viologen, pH 9.0), 19 (F420, pH 7.2), 34 (Fo, 7.8-didemethyl-8-hydroxy-5-deazariboflavin, pH 7.2), 10 (H2, Fo as co-substrate, pH 7.2), 2 (H2, methyl viologen as co-substrate, pH 9.0). The native protein is oligomeric (apparent Mr greater than 500,000) and is composed of three distinct subunits with Mr - 40,000, 31,000, and 26,000 in the ratio of 2:2:1, leading to a minimum Mr = 170,000. In addition to 33 atoms of Fe and 24 atoms of acid-labile sulfur, the F420-hydrogenase contains 2.3 mol of FAD/mol of Mr - 170,000. This activity is chromatographically distinct from a smaller methanogen hydrogenase capable of rapid viologen reduction, but which only very slowly reduces 5-deazariboflavins.