Web Release Date: February 16,
Characterization of a Succinyl-CoA Radical-Cob(II)alamin Spin Triplet
Intermediate in the Reaction Catalyzed by Adenosylcobalamin-Dependent
Methylmalonyl-CoA Mutase
and
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53726-4087, and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664
Received August 18, 2004
Revised Manuscript Received December 15, 2004
Abstract:
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 
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.
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