Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate

Xianghui Li, Joshua Telser, Ryan C. Kunz, Brian M. Hoffman, Gary J. Gerfen, Stephen W. Ragsdale

Research output: Contribution to journalArticle

14 Citations (Scopus)

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)-F430 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, 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)-MCRred1 state of the enzyme decays. Subsequently, a radical (MCRBES radical) 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 MCRBES 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 MCRBES 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.

Original languageEnglish (US)
Pages (from-to)6866-6876
Number of pages11
JournalBiochemistry
Volume49
Issue number32
DOIs
StatePublished - Aug 17 2010

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Electron Spin Resonance Spectroscopy
Organometallics
Observation
Phosphothreonine
Paramagnetic resonance
Methane
Spectrum Analysis
Spectroscopy
Kinetics
Electrons
Archaea
Prosthetics
Protons
Substitution reactions
Monomers
methyl coenzyme M reductase
Enzymes
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate. / Li, Xianghui; Telser, Joshua; Kunz, Ryan C.; Hoffman, Brian M.; Gerfen, Gary J.; Ragsdale, Stephen W.

In: Biochemistry, Vol. 49, No. 32, 17.08.2010, p. 6866-6876.

Research output: Contribution to journalArticle

Li, Xianghui ; Telser, Joshua ; Kunz, Ryan C. ; Hoffman, Brian M. ; Gerfen, Gary J. ; Ragsdale, Stephen W. / Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate. In: Biochemistry. 2010 ; Vol. 49, No. 32. pp. 6866-6876.
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T1 - Observation of organometallic and radical intermediates formed during the reaction of methyl-coenzyme M reductase with bromoethanesulfonate

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AU - Gerfen, Gary J.

AU - Ragsdale, Stephen W.

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N2 - 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)-F430 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, 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)-MCRred1 state of the enzyme decays. Subsequently, a radical (MCRBES radical) 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 MCRBES 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 MCRBES 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.

AB - 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)-F430 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, 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)-MCRred1 state of the enzyme decays. Subsequently, a radical (MCRBES radical) 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 MCRBES 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 MCRBES 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.

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