On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F 430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 Å and 1.8 Å resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F 430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg 2+ and 9 Na + ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 Å resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.