BIOCHEMICAL-CHARACTERIZATION OF THE 8-HYDROXY-5-DEAZAFLAVIN-REACTIVE HYDROGENASE FROM METHANOSARCINA-BARKERI FUSARO

The membrane-associated coenzyme F-420-reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95-fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F-420-reducing activity (82.8 mu mol . min(-1) . mg protein(-1) versus 11.5 mu mol . min(-1) . mg protein(-1)) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79-88]. The predominant coenzyme F-420-reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non-identical subunits with apparent molecular masses of 48 (alpha), 33 (beta), and 30 kDa (gamma), apparently in a stoichiometry of alpha(2) beta(2) gamma(1). This minimal coenzyme F-420-reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198-kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28-32 mol non-heme iron, and 34 mol acid-labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N-termini of the putative nickel-binding subunits of most [NiFe]- and [NiFeSe]hydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F-420-reducing activity was obtained with reductively reactivated enzyme at 55 degrees C in the pH range 6.5-7.25. The K-m values of the purified enzyme for H-2 with coenzyme F-420 or methylviologen as electron acceptor were extremely low, namely 3 mu M and 4 mu M. The catalytic efficiency coefficients (k(cat)/K-m) for H-2 with both reducible cosubstrates were high: 2.5x10(7) M(-1) . s(-1) with coenzyme F-420 and 6.9X10(7) M(-1) . s(-1) with methylviologen.