The Elusive 5′-Deoxyadenosyl Radical in Coenzyme-B₁₂-Mediated Reactions

Abstract

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12) (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(•)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

“…Other AdoCbl-dependent mutases similarly position their substrates at the same distance from the Cbl, as determined from crystal structures (24,29,33) and by electron paramagnetic resonance spectroscopic studies of glutamate mutase (62) and MCM (63) under catalytic conditions. Pseudorotation of the 5Ј-dAdo ribose has also been suggested from structural and biochemical studies of glutamate mutase (33,55) and from computational studies on MCM (64). Thus, given the combined structural, biochemical, and computational evidence, it appears that this mechanism of moving the active C5Ј radical toward substrate is conserved in AdoCbl-dependent mutases such as acyl-CoA mutases.…”

The entropic contributions in vitamin B 12 enzymes still reflect the electrostatic paradigm

“…In further elaborating on the state of current theoretical studies, we would like to clarify that even the technically impressive (by the size of the QM region) results of the MTD study of MCM (17) has not been as reliable as one might tend to think (SI Text). Here again, the conclusion (17) that the surface cannot be concerted is problematic, as the actual calculated surface is quite flat in its diagonal range.…”

“…8) of using very careful QM calculations in solution (with a higher-level QM model than that used in ref. 17) have produced a concerted path, and that moving the solution surface to the protein environment by a calibrated EVB model is expected to be more reliable (both in terms of the sampling and in terms of extrapolation of reliable reference systems) than the direct MTD in the protein site (see SI Text for more discussion). Finally, the problems with the MTD surface of ref.…”

“…17 also include the fact that the low level used does not produce correct radical transfer energies (as established in fact by other careful works of the authors of ref. 17).…”

“…Unfortunately, the reliability and the nature of the EVB calculations have not been fully appreciated as is apparent from a subsequent metadynamics (MTD) study (17) that will be addressed below. Furthermore, the fact that the EVB calculations have evaluated the total activation free energy rather than some enthalpy contribution has not been appreciated, or was simply overlooked.…”