Methyl-coenzyme M reductase from methanogenic archaea: Isotope effects on label exchange and ethane formation with the homologous substrate ethyl-coenzyme M

Silvan Scheller, Meike Goenrich, Rudolf K. Thauer, Bernhard Jaun*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

16 Citations (Scopus)

Abstract

Ethyl-coenzyme M (CH3CH2-S-CH2CH 2-SO3 -, Et-S-CoM) serves as a homologous substrate for the enzyme methyl-coenzyme M reductase (MCR) resulting in the product ethane instead of methane. The catalytic reaction proceeds via an intermediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-limiting step, many cycles of intermediate formation and reconversion to substrate occur before a substantial amount of ethane is released. In deuterated buffer, the intermediate becomes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables intermediate formation to be detected. Here, we present a comprehensive analysis of this pre-equilibrium. 2H- and 13C-labeled isotopologues of Et-S-CoM were used as the substrates, and the time course of each isotopologue was followed by NMR spectroscopy. A kinetic simulation including kinetic isotope effects allowed determination of the primary and α- and β-secondary isotope effects for intermediate formation and for the C-H/C-D bond activation in the ethane-containing intermediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. Am. Chem. Soc. 2013, 135, DOI: 10.1021/ja406485z10.1021/ja406485z) and thus imply the same catalytic mechanism for both substrates. The experiment by Floss and co-workers, demonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is compatible with the observed rapid isotope exchange if the isotope effects measured here are taken into account.

Original languageEnglish
Pages (from-to)14985-14995
Number of pages11
JournalJournal of the American Chemical Society
Volume135
Issue number40
DOIs
Publication statusPublished - 2013
MoE publication typeA1 Journal article-refereed

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