Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum.

摘要: Abstract The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights 43,000, 36,700, 28,800, respectively) formed aggregates weight, 1,020,000) a F420-active alpha 1 beta trimer 109,000). mol flavin adenine dinucleotide (FAD), nickel, 12 14 iron, 11 acid-labile sulfide per the 109,000-molecular-weight species, but no selenium. isoelectric point 5.6. amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any acid) conserved in N-termini F420-hydrogenases from M. thermoautotrophicum largest nickel-containing hydrogenases Desulfovibrio baculatus, gigas, Rhodobacter capsulatus. F420-hydrogenase required reductive reactivation before assay. FAD dissociated during unless potassium salts were present, yielding deflavoenzyme that unable reduce F420. Maximal activity obtained at 55 degrees C pH 7.0 7.5, with 0.2 0.8 M KCl reaction mixture. catalyzed H2 production rate threefold lower than for uptake reduced F420, methyl viologen, flavins, 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited F420-dependent not viologen-dependent enzyme.