Uncovering Major Structural and Functional Features of Methyl-Coenzyme M Reductase (MCR) from Methanobrevibacter ruminantium in Complex with Two Substrates

Structural insights into methyl-coenzyme M reductase from <i>Methanobrevibacter ruminantium</i> (<i>M. ruminantium</i>) has implications for methane mitigation strategies. Methanogenesis in ruminants is a major contributor to global greenhouse gas emissions, primarily driven by the rumen archaeon <i>M. ruminantium</i>. Central to this process is methyl-coenzyme M reductase (Mcr), an enzyme that catalyzes the final step of methane production. Despite its significance as a chemogenetic target for methane mitigation, the high-resolution structure of <i>M. ruminantium</i> Mcr has remained elusive. Here, we employed homology modeling and CDOCKER simulations within the CHARMM force field to elucidate the structural and functional features of the <i>M. ruminantium</i> Mcr/ligand complexes. We characterized two distinct states: the reduced Mcr_oxi-silent state bound to HS-CoM and CoB-SH, and the oxidized Mcr_silent state bound to the heterodisulfide CoM-S-S-CoB. Alanine-scanning mutagenesis identified 71 and 62 key residues per active site for each state, respectively, revealing the fundamental determinants of structural stability and substrate selectivity on the Ni-F₄₃₀ cofactor. Furthermore, structure-based pharmacophore modeling defined essential features (AAADDNNN and AAADDNN) that drive ligand binding. These findings provide a high-resolution molecular framework for the rational design of specific Mcr inhibitors, offering a robust starting point for developing broad-spectrum strategies to suppress enteric methane emissions.