Evidence for a 1,2 Shift of a Hydrogen Atom in a Radical Intermediate of the Methylmalonyl-CoA Mutase Reaction

Abstract

An excellent substrate of methylmalonyl-CoA mutase, methylmalonyl-carba-(dethia) coenzyme A (methylmalonyl-CH2-CoA), was synthesized by a chemoenzymatic method and its α-proton was exchanged with deuterium by long-term incubation in deuterium oxide at pH 6.9. After addition of highly purified epimerase-free methylmalonyl-CoA mutase the enzymatic rearrangement was monitored by 1H NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH2-CoA was monodeuterated, while unlabeled and geminally dideuterated species, 14% of each, were also formed. After the addition of more enzyme the equilibrium (methylmalonyl-CoA:succinyl-CoA = 1:20) was quickly established, while the proportion of unlabeled succinyl-CH2-CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ratio of the unlabeled, monodeuterated, and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurred. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH2-CoA radical intermediate competing with the hydrogen transfer from 5′-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations.