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

The membrane-associated coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained α, β, and γ subunits (molecular weights of 43,600, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F₄₂₀-active α₁β₁γ₁ trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N₃-P-N₂-R-N₁-EGH-N₆-V (where N is any amino acid) was conserved in the N-termini of the α subunits of the F₄₂₀-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F₄₂₀-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F₄₂₀. Maximal coenzyme F₄₂₀-reducing activity was obtained at 55°C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H₂ production at a rate threefold lower than that for H₂ uptake and reduced coenzyme F₄₂₀, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5'deazariboflavin. Specific antiserum inhibited the coenzyme F₄₂₀-dependent but not the methyl viologen-dependent activity of the purified enzyme.