Continuous wave photolysis magnetic field effect investigations with free and protein-bound alkylcobalamins

The activation of the Co-C bond in adenosylcobalamin-dependent enzymes generates a singlet-born Co(II)-adenosyl radical pair. Two of the salient questions regarding this process are: (1) What is the origin of the considerable homolysis rate enhancement achieved by this class of enzyme? (2) Are the reaction dynamics of the resultant radical pair sensitive to the application of external magnetic fields? Here, we present continuous wave photolysis magnetic field effect (MFE) data that reveal the ethanolamine ammonia lyase (EAL) active site to be an ideal microreactor in which to observe enhanced magnetic field sensitivity in the adenosylcobalamin radical pair. The observed field dependence is in excellent agreement with that calculated from published hyperfine couplings for the constituent radicals, and the magnitude of the MFE (<18%) is almost identical to that observed in a solvent containing 67% glycerol. Similar augmentation is not observed, however, in the equivalent experiments with EAL-bound methylcobalamin, where all field sensitivity observed in the free cofactor is washed out completely. Parallels are drawn between the latter case and the loss of field sensitivity in the EAL holoenzyme upon substrate binding (Jones et al. J. Am. Chem. Soc. 2007, 129, 15718-15727). Both are attributed to the rapid removal of the alkyl radical immediately after homolysis, such that there is inadequate radical pair recombination for the observation of field effects. Taken together, these results support the notion that rapid radical quenching, through the coupling of homolysis and hydrogen abstraction steps, and subsequent radical pair stabilization make a contribution to the observed rate acceleration of Co-C bond homolysis in adenosylcobalamin-dependent enzymes.