TIME-RESOLVED SPECTROSCOPIC STUDIES OF B-12 COENZYMES, THE IDENTIFICATION OF A METASTABLE COB(III)ALAMIN PHOTOPRODUCT IN THE PHOTOLYSIS OF METHYLCOBALAMIN

Ultrafast transient absorption spectroscopy has been used to investiga te the primary photochemistry of methylcobalamin. Approximately 27% of the initially excited methylcobalamin undergoes a bond homolysis on a subpicosecond time scale. The remaining 73% forms a metastable photop roduct with a spectrum similar to that of cob(III)alamin compounds. Th e ultraviolet absorption spectrum of the metastable photoproduct exhib its a prominent gamma-band at 340 nm, characteristic of a cob(III)alam in with a very weak axial ligand. The metastable photoproduct recovers to the ground electronic state of methylcobalamin on a 1.2 +/- 0.5 ns time scale, leaving only cob(II)alamin (and presumably methyl radical ) at 9 ns. The primary photochemical yield of cob(II)alamin is determi ned largely by the branching ratio between the two photoproduct channe ls. A 40 ps transient absorption difference spectrum of methylcobalami n bound to methionine synthase indicates that the branching ratio and initial production of cob(II)alamin is not changed in the enzyme-bound cofactor. The substantial photolysis protection afforded by the enzym e must be attributed to structural and electronic effects which enhanc e the intrinsic rate of recombination of the radical pair, rather than to suppression of primary bond homolysis.