title: Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum.
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 of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 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 alpha 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 degrees 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.
authors.affiliation: Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: S F
authors.initials: SF
authors.lastname: Baron
authors.forename: J G
authors.initials: JG
authors.lastname: Ferry
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019605:Euryarchaeota / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000502:physiology; D007106:Immune Sera / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008670:Metals / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007106:Immune Sera; D008565:Membrane Proteins; D008670:Metals; D005182:Flavin-Adenine Dinucleotide; C015084:coenzyme F420; D010088:Oxidoreductases; C033895:coenzyme F420 hydrogenase; D012256:Riboflavin