Isotopic Evidence for Reaction Pathways and Quantum Tunneling in Methanotrophy

The isotope composition of residual methane after microbial oxidation can provide rigorous constraints on proposed methanotrophic pathways. However, mechanistic interpretation of the abundances of naturally occurring partially substituted methane isotopomers requires accurate kinetic isotope effects (KIEs) obtained from first-principles computations, followed by kinetic calculations specific to each isotopomer. Here, we apply contemporary density functional theory methods to calculate KIEs including the quantum tunneling effects and subsequently predict kinetic isotope fractionation involving various isotopic substitutions for several possible aerobic and anaerobic methane oxidation pathways. The results successively reproduced the abundances of methane isotopomers (13CH4, 12CH3D, 13CH3D, and 12CH2D2) reported in recent experimental studies. For aerobic methanotrophy catalyzed by copper-dependent enzymes, we identify the antiferromagnetic bis(μ-oxo)dicopper(II) complex as an active oxidant that exhibits substantial quantum tunneling. This pathway quantitatively reproduces not only the large D fractionation but also the large 13C fractionation and the pronounced depletion (relative to stochastic abundances) of 13CH3D and CH2D2 in residual methane observed in laboratory experiments conducted under excess copper conditions. In contrast, the mixed-valence Cu(II)+Cu(III) dicopper-dioxo complex exhibits negligible quantum tunneling and fails to reproduce the observed isotopic signatures. For anaerobic methanotrophy, the computed KIEs support methane activation by the sulfur-centered radical on coenzyme B rather than by the nickel center of the F430 cofactor. This mechanism reproduces the modest isotope effects observed experimentally and exhibits minimal quantum tunneling contributions. Overall, this work presents a computational workflow to distinguish competing reaction pathways using the isotopic compositions of multiple isotopologues in complex microbial systems under natural or engineered conditions. The results further demonstrate that the modern quantum chemical methods are sufficiently advanced to elucidate biochemical reaction mechanisms from isotope effects without requiring comprehensive isotope labeling.