Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate.
Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate.
复制标题
甲基辅酶 M 还原酶与溴乙磺酸盐反应过程中形成的有机金属和自由基中间体的观察。
DOI：
10.1021/bi100650m
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Ragsdale,StephenW
中科院分区：
文献类型：
--
作者：
Li,Xianghui;Telser,Joshua;Kunz,RyanC;Hoffman,BrianM;Gerfen,Gary;Ragsdale,StephenW
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)-F430prosthetic groups, one in each monomer. This report describes studies of the reaction of the active Ni(I) state of MCR (MCRred1) with BES (2-bromoethanesulfonate) and CoBSH or its analogue, CoB6SH (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)-MCRred1state of the enzyme decays. Subsequently, a radical (MCRBESradical) is formed that was characterized by multifrequency electron paramagnetic resonance (EPR) studies at X- (∼9 GHz), Q- (∼35 GHz), and D- (∼130 GHz) bands and by electron−nuclear double resonance (ENDOR) spectroscopy. The MCRBESradical is characterized byg-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 MCRBESis 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.
