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Abstract

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCRred1 both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCRred1 signal is partially converted into the rhombic EPR signal MCRred2. To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic 1H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCRred2 state.