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 (CH(3)S-CH(2)CH(2)SO(3)) 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 gamma-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 (14)N 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.
Telser
J
J
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, USA.
Davydov
R
R
Horng
Y C
YC
Ragsdale
S W
SW
Hoffman
B M
BM
D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism; D017012:Methanobacteriales / Q000201:enzymology*; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*
D003067:Coenzymes; D008665:Metalloporphyrins; C023540:factor F430; D009532:Nickel; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase