title: Methyl-coenzyme M reductase preparations with high specific activity from H2-preincubated cells of Methanobacterium thermoautotrophicum.
abstract: The study of the nickel enzyme methyl-coenzyme M reductase from methanogenic bacteria has been hampered until now by the fact that upon cell rupture the activity of the enzyme always dropped to at best only a few percent of its in vivo activity. We describe here that when Methanobacterium thermoautotrophicum cells were preincubated with 100% H2 before disintegration methyl-coenzyme M reductase activity stayed high. The cell extracts with a specific activity of 2 U/mg protein exhibited two nickel-derived EPR signals, designated MCR-red1 and MCR-red2, previously only observed in intact cells. The enzyme was purified 10-fold to a specific activity of 20 U/mg in the presence of methyl-coenzyme M, which stabilized both the activity and the EPR signal MCR-red1. The enzyme preparation displayed an UV/Vis spectrum with an absorption maximum at 386 nm and a shoulder at 420 nm. Upon inactivation of the enzyme with O2 or CHCl3, the maximum at 386 nm and the EPR signals MCR-red1 and MCR-red2 disappeared.
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität Marburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Rospert
authors.forename: R
authors.initials: R
authors.lastname: Böcher
authors.forename: S P
authors.initials: SP
authors.lastname: Albracht
authors.forename: R K
authors.initials: RK
authors.lastname: Thauer
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006859:Hydrogen / Q000494:pharmacology*; D017014:Methanobacterium / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D006859:Hydrogen; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase