Cryoreduction of methyl-coenzyme M reductase: EPR characterization of forms, MCRox1 and MCRred1

J. Telser, R. Davydov, Y. C. Horng, S. W. Ragsdale, B. M. Hoffman

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Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methyl-coenzyme M (CH3SCH2CH2SO3) from methane. The active site is a nickel tetrahydrocorphinoid cofactor, factor 430, which in inactive form contains EPR-silent Ni(II). Two such forms, denoted MCRsilent and MCRox1-silent, were previously structurally characterized by X-ray crystallography. We describe here the cryoreduction of both of these MCR forms by γ-irradiation at 77 K, which yields reduced protein maintaining the structure of the oxidized starting material. Cryoreduction of MCRsilent yields an EPR signal that strongly resembles that of MCRred1, the active form of MCR; and stepwise annealing to 260-270 K leads to formation of MCRred1. Cryoreduction of MCRox1-silent solutions shows that our preparative method for this state yields enzyme that contains two major forms. One behaves similarly to MCRsilent, as shown by the observation that both of these forms give essentially the same redlike EPR signals upon cryoreduction, both of which give MCRred1 upon annealing. The other form is assigned to the crystallographically characterized MCRox1-silent and directly gives MCRox1 upon cryoreduction. X-band spectra of these cryoreduced samples, and of conventionally prepared MCRred1 and MCRox1, all show resolved hyperfine splitting from four equivalent nitrogen ligands with coupling constants in agreement with those determined in previous EPR studies and from 14N ENDOR of MCRred1 and MCRox1. These experiments have confirmed that all EPR-visible forms of MCR contain Ni(I) and for the first time generated in vitro the EPR-visible, enzymatically active MCRred1 and the activate-able "ready" MCRox1 from "silent" precursors. Because the solution Ni(II) species we assign as MCRox1-silent gives as its primary cryoreduction product the Ni(I) state MCRox1, previous crystallographic data on MCRox1-silent allow us to identify the exogenous axial ligand in MCRox1 as the thiolate from CoM; the cryoreduction experiments further allow us to propose possible axial ligands in MCRred1. The availability of model compounds for MCRred1 and MCRox1 also is discussed.

Original languageEnglish
Pages (from-to)5853-5860
Number of pages8
JournalJournal of the American Chemical Society
Volume123
Issue number25
DOIs
Publication statusPublished - 2001 Oct 3

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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