Cryoreduction of methyl-coenzyme M reductase: EPR characterization of forms, MCR_ox1 and MCR_red1

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 MCR_silent and MCR_ox1-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 MCR_silent yields an EPR signal that strongly resembles that of MCR_red1, the active form of MCR; and stepwise annealing to 260-270 K leads to formation of MCR_red1. Cryoreduction of MCR_ox1-silent solutions shows that our preparative method for this state yields enzyme that contains two major forms. One behaves similarly to MCR_silent, as shown by the observation that both of these forms give essentially the same redlike EPR signals upon cryoreduction, both of which give MCR_red1 upon annealing. The other form is assigned to the crystallographically characterized MCR_ox1-silent and directly gives MCR_ox1 upon cryoreduction. X-band spectra of these cryoreduced samples, and of conventionally prepared MCR_red1 and MCR_ox1, 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 MCR_red1 and MCR_ox1. 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 MCR_red1 and the activate-able "ready" MCR_ox1 from "silent" precursors. Because the solution Ni(II) species we assign as MCR_ox1-silent gives as its primary cryoreduction product the Ni(I) state MCR_ox1, previous crystallographic data on MCR_ox1-silent allow us to identify the exogenous axial ligand in MCR_ox1 as the thiolate from CoM; the cryoreduction experiments further allow us to propose possible axial ligands in MCR_red1. The availability of model compounds for MCR_red1 and MCR_ox1 also is discussed.