Characterization of the thioether product formed from the thiolytic cleavage of the alkyl-nickel bond in methyl-coenzyme M reductase.

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in methanogenesis by using N-7-mercaptoheptanolyl-threonine phosphate (CoBSH) as the two-electron donor to reduce 2-(methylthiol)ethane sulfonate (methyl-SCoM) to methane, and producing the heterodisulfide, CoBS-SCoM. The active site of MCR includes a noncovalently bound Ni tetrapyrrolic cofactor called coenzyme F430, which is in the Ni(I) state in the active enzyme (MCRred1). Bromopropanesulfonate (BPS) is a potent inhibitor and reversible redox inactivator that reacts with MCRred1 to form an EPR-active state called MCRPS, which is an alkyl-nickel species. When MCRPS is treated with free thiol containing compounds, the enzyme is reconverted to the active MCRred1 state. In this paper, we demonstrate that the reactivation of MCRPS to MCRred1 by thiols involves formation of a thioether product. MCRPS also can be converted to active MCRred1 by treatment with sodium borohydride. Reactivation is highest with the smallest free thiol HS-. Interestingly, MCRPS can also be reductively activated with analogues of CoBSH such as CoB8SH and CoB9SH, but not CoBSH itself. Unambiguous demonstration of the formation of a methylthioether product from thiolysis of an alkyl-Ni species provides support for a methyl-Ni intermediate in the MCR-catalyzed last step in methanogenesis and the first proposed step in anaerobic methane oxidation.