Methyl-coenzyme M reductase preparations with high specific activity from H2-preincubated cells of Methanobacterium thermoautotrophicum.
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.
Rospert
S
S
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität Marburg, Germany.
Böcher
R
R
Albracht
S P
SP
Thauer
R K
RK
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
D006859:Hydrogen; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase