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Abstract

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F430 as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K M=20 mM; apparent V max=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K M=5 mM; apparent V max=30 U/mg). Propyl-coenzyme M (apparent K i=2 mM) and allyl-coenzyme M (apparent K i=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I]0.5 V=2 µM), cyano-coenzyme M ([I]0.5 V=0.2 mM), 3-bromopropionate ([I]0.5 V=3 mM), seleno-coenzyme M ([I]0.5 V=6 mM) and trifluoromethyl-coenzyme M ([I]0.5 V=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D2O. The signal was abolished upon exposure of the enzyme to O2. 3-Bromopropanesulfonate ([I]0.5 V=0.1 µM), 3-iodopropanesulfonate ([I]0.5 V=1 µM), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O2. 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.