Characterization of a Succinyl-CoA Radical−Cob(II)alamin Spin Triplet Intermediate in the Reaction Catalyzed by Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase^†

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron−electron spin coupling between an organic radical and the low-spin Co²⁺ in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co²⁺. Labeling of the substrate with ¹³C and ²H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2‘-¹³C]methylmalonyl-CoA and [2-¹³C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon α to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.