title: Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate.
abstract: Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step of methane formation, in which methyl-coenzyme M (2-methylthioethanesulfonate, methyl-SCoM) is reduced with coenzyme B (N-(7-mercaptoheptanoyl)threonine phosphate, CoBSH) to form methane and the heterodisulfide CoBS-SCoM. The active dimeric form of MCR contains two Ni(I)-F(430) prosthetic groups, one in each monomer. This report describes studies of the reaction of the active Ni(I) state of MCR (MCR(red1)) with BES (2-bromoethanesulfonate) and CoBSH or its analogue, CoB(6)SH (N-(6-mercaptohexanoyl)threonine phosphate), by transient kinetic measurements using EPR and UV-visible spectroscopy and by global fits of the data. This reaction is shown to lead to the formation of three intermediates, the first of which is assigned as an alkyl-Ni(III) species that forms as the active Ni(I)-MCR(red1) state of the enzyme decays. Subsequently, a radical (MCR(BES) radical) is formed that was characterized by multifrequency electron paramagnetic resonance (EPR) studies at X- ( approximately 9 GHz), Q- ( approximately 35 GHz), and D- ( approximately 130 GHz) bands and by electron-nuclear double resonance (ENDOR) spectroscopy. The MCR(BES) radical is characterized by g-values at 2.00340 and 1.99832 and includes a strongly coupled nonexchangeable proton with a hyperfine coupling constant of 50 MHz. Based on transient kinetic measurements, the formation and decay of the radical coincide with a species that exhibits absorption peaks at 426 and 575 nm. Isotopic substitution, multifrequency EPR, and ENDOR spectroscopic experiments rule out the possibility that MCR(BES) is a tyrosyl radical and indicate that if a tyrosyl radical is formed during the reaction, it does not accumulate to detectable levels. The results provide support for a hybrid mechanism of methanogenesis by MCR that includes both alkyl-Ni and radical intermediates.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Xianghui
authors.initials: X
authors.lastname: Li
authors.forename: Joshua
authors.initials: J
authors.lastname: Telser
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Kunz
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Gary
authors.initials: G
authors.lastname: Gerfen
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D017738:Alkanesulfonic Acids / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D017013:Methanobacteriaceae / Q000737:chemistry* / Q000201:enzymology; D008956:Models, Chemical; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: D017738:Alkanesulfonic Acids; C018853:2-bromoethanesulfonic acid; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase