Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.

Methyl-coenzyme M reductase (MCR) is the key enzyme in methane formation by methanogenic Archaea. It converts the thioether methyl-coenzyme M and the thiol coenzyme B into methane and the heterodisulfide of coenzyme M and coenzyme B. The catalytic mechanism of MCR and the role of its prosthetic group, the nickel hydrocorphin coenzyme F(430), is still disputed, and no intermediates have been observed so far by fast spectroscopic techniques when the enzyme was incubated with the natural substrates. In the presence of the competitive inhibitor coenzyme M instead of methyl-coenzyme M, addition of coenzyme B to the active Ni(I) state MCR(red1) induces two new species called MCR(red2a) and MCR(red2r) which have been characterized by pulse EPR spectroscopy. Here we show that the two MCR(red2) signals can also be induced by the S-methyl- and the S-trifluoromethyl analogs of coenzyme B. (19)F-ENDOR data for MCR(red2a) and MCR(red2r) induced by S-CF(3)-coenzyme B show that, upon binding of the coenzyme B analog, the end of the 7-thioheptanoyl chain of coenzyme B moves closer to the nickel center of F(430) by more than 2 A as compared to its position in both, the Ni(I) MCR(red1) form and the X-ray structure of the inactive Ni(II) MCR(ox1-silent) form. The finding that the protein is able to undergo a conformational change upon binding of the second substrate helps to explain the dramatic change in the coordination environment induced in the transition from MCR(red1) to MCR(red2) forms and opens the possibility that nickel coordination geometries other than square planar, tetragonal pyramidal, or elongated octahedral might occur in intermediates of the catalytic cycle.