Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F₄₃₀ Modifications

Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F₄₃₀, a nickel-containing tetrahydrocorphin. Several modified versions of F₄₃₀ have been discovered, including the 17²-methylthio-F₄₃₀ (mtF₄₃₀) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F₄₃₀ compared to 17²-thioether coenzyme F₄₃₀ variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 17² methylthio modification in ANME-1 MCR. Modifications at the 17² position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F₄₃₀ variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH₃-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme's organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.