Abstract
The complex of dioldehydrase with adenosylcobalamin (coenzyme B12) and potassium ion reacts with molecular oxygen in the absence of a substrate to oxidize coenzyme and inactivate the complex. In this article, high performance liquid chromatography and mass spectral analysis are used to identify the nucleoside products resulting from oxygen inactivation. The product profile indicates that oxygen inactivation proceeds by direct reaction of molecular oxygen with the 5'-deoxyadenosyl radical and cob(II)alamin. Formation of 5'-peroxyadenosine as the initial nucleoside product chemically correlates this reaction with aerobic, aqueous photoinduced homolytic cleavage of adenosylcobalamin (Schwartz, P. A., and Frey, P. A., (2007) Biochemistry, in press), indicating that both reactions proceed through similar chemical intermediates. The oxygen inactivation of the enzyme-coenzyme complex shows an absolute requirement for the same monocations required in catalysis by dioldehydrase. Measurements of the dissociation constants for adenosylcobalamin from potassium-free (Kd = 16 +/- 2 microM) or potassium-bound dioldehydrase (Kd = 0.8 +/- 0.2 microM) reveal that the effect of the monocation in stimulating oxygen sensitivity cannot be explained by an effect on the binding of coenzyme to the enzyme. Cross-linking experiments suggest that the full quaternary structure is assembled in the absence of potassium ion under the experimental conditions. The results indicate that dioldehydrase likely harvests the binding energy of the activating monocation to stimulate the homolytic cleavage of the Co-C5' bond in adenosylcobalamin.