Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro.
The membrane-associated coenzyme F420-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 F420-reducing activity (82.8 mumol.min-1.mg protein-1 versus 11.5 mumol.min-1.mg protein-1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79-88]. The predominant coenzyme F420-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 F420-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 F420-reducing activity was obtained with reductively reactivated enzyme at 55 degrees C in the pH range 6.5-7.25. The Km values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3 microM and 4 microM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5 x 10(7) M-1.s-1 with coenzyme F420 and 6.9 x 10(7) M-1.s-1 with methylviologen.
Michel
R
R
Institut für Pflanzenphysiologie und Mikrobiologie, Freien Universität Berlin, Germany.
Massanz
C
C
Kostka
S
S
Richter
M
M
Fiebig
K
K
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D017021:Methanosarcina barkeri / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis
C007701:factor 420; D010088:Oxidoreductases; C033895:coenzyme F420 hydrogenase; D012256:Riboflavin