Femtosecond to nanosecond transient absorption spectroscopy has been used to investigate the primary photochemistry of the B 12 coenzymes, methylcobalamin and 5′-deoxyadenosylcobalamin. Photolysis at excitation wavelengths in the near UV (400 nm) and visible (520-530 nm) are compared. Measurements were performed with femtosecond time resolution covering time delays of up to 9 ns. The photochemistry of methylcobalamin is found to depend strongly on excitation wavelength, while the photochemistry of adenosylcobalamin is essentially wavelength independent over the range studied. Excitation of methylcobalamin at 400 nm results in a partitioning between prompt bond homolysis and formation of a metastable cob(III)-alamin photoproduct as reported earlier [Walker, L. A., II; Jarrett, J. T.; Anderson, N. A.; Pullen, S. H.; Matthews, R. G.; Sension, R. J. J. Am. Chem. Soc. 1998, 120, 3597-3603]. Excitation of methylcobalamin at 520 nm in the visible R -band results only in formation of the metastable cob(III)alamin photoproduct. No prompt bond homolysis is observed. The metastable photoproduct partitions between formation of cob(II)-alamin (14 ( 5%) and recovery of the methylcobalamin starting material (86 ( 5%) on a 1.0 ( 0.1 ns time scale. The quantum yield for bond homolysis in methylcobalamin is determined by the wavelength-dependent partitioning between prompt homolysis and formation of the metastable photoproduct and by the partitioning of the metastable photoproduct between bond homolysis and ground-state recovery. In contrast, excitation of adenosylcobalamin at both 400 and 520 nm results in the development of a difference spectrum characteristic of the formation of cob(II)alamin on a picosecond time scale. The quantum yield for bond homolysis in this case is determined primarily by the competition between geminate recombination and diffusion to form solventseparated radical pairs. The caging fraction for adenosylcobalamin in aqueous solution at room temperature is 0.71 ( 0.05.

An ultrafast transient absorption study of the primary photolysis of ethyl- and n-propylcobalamin in water is presented. Data have been obtained for two distinct excitation wavelengths, 400 nm at the edge of the UV gamma-band absorption, and 520 nm in the strong visible alphabeta-band absorption. These data are compared with results reported earlier for the B(12) coenzymes, methyl- and adenosylcobalamin. The data obtained for ethylcobalamin and n-propylcobalamin following excitation at 400 nm demonstrate the formation of one major photoproduct on a picosecond time scale. This photoproduct is spectroscopically identifiable as a cob(II)alamin species. Excitation of methyl-, ethyl-, and n-propylcobalamin at 520 nm in the low-lying alphabeta absorption band results in bond homolysis proceeding via a bound cob(III)alamin MLCT state. For all of the cobalamins studied here competition between geminate recombination of caged radical pairs and cage escape occurs on a time scale of 500 to 700 ps. The rate constants for geminate recombination in aqueous solution fall within a factor of 2 between 0.76 and 1.4 ns(-1). Intrinsic cage escape occurs on time scales ranging from

Femtosecond transient absorption spectroscopy has been used to investigate the photolysis of coenzyme B12 (5‘-deoxyadenosylcobalamin). Transient kinetic measurements obtained at wavelengths between 400 and 633 nm were analyzed globally to obtain time constants. Transient spectral data obtained for time delays between 5 ps and 9 ns were analyzed by matrix decomposition to identify distinct spectral components present in the data. Photoexcitation results in homolysis of the carbon−cobalt bond forming a singlet radical pair on a picosecond time scale. The subsequent spectral changes probe conformational relaxation and geminate recombination. Analysis of the spectral data suggests that 76 ± 4% of the geminate radical pairs recombine, resulting in a quantum yield of 0.24 ± 0.04 for the formation of solvent separated radicals, in good agreement with literature values of 0.20 ± 0.03 and 0.23 ± 0.04 [Chen, E.; Chance, M. R. Biochemistry 1993, 32, 1480−1487]. The geminate recombination of the adenosyl radical with cob(II)alamin occurs biphasically with exponential time constants of 150 ± 20 ps and 0.5 ± 0.2 ns. The effective recombination rate from a single-exponential fit to the data is (0.250 ns)-1 = 4 ns-1.

Femtosecond transient IR and visible absorption spectroscopies have been employed to investigate the excited-state photophysics of vitamin B12 (cyanocobalamin, CNCbl) and the related cob(III)alamins, azidocobalamin (N3Cbl), and aquocobalamin (H2OCbl). Excitation of CNCbl, H2OCbl, or N3Cbl results in rapid formation of a short-lived excited state followed by ground-state recovery on time scales ranging from a few picoseconds to a few tens of picoseconds. The lifetime of the intermediate state is influenced by the sigma-donating ability of the axial ligand, decreasing in the order CNCbl > N3Cbl > H2OCbl, and by the polarity of the solvent, decreasing with increasing solvent polarity. The peak of the excited-state visible absorption spectrum is shifted to ca. 490 nm, and the shape of the spectrum is characteristic of weak axial ligands, similar to those observed for cob(II)alamin, base-off cobalamins, or cobinamides. Transient IR spectra of the upper CN and N3 ligands are red-shifted 20-30 cm(-1) from the ground-state frequencies, consistent with a weakened Co-upper ligand bond. These results suggest that the transient intermediate state can be attributed to a corrin ring pi to Co 3d(z2) ligand to metal charge transfer (LMCT) state. In this state bonds between the cobalt and the axial ligands are weakened and lengthened with respect to the corresponding ground states.