Ultrafast transient absorption studies of coenzyme B12 analogs and derivatives: Biological activity of alkylcobalamins

Ultrafast spectroscopic techniques have been employed to observe the photolytic bond cleavage of the carbon-cobalt bond in B₁₂ coenzymes and derivatives. The various systems studied include: 5′-deoxyadenosyl-, methyl-, ethyl- and n-propylcobalamin. Studies were performed under anaerobic conditions in both an aqueous environment as well as an ethylene glycol environment. For the pump-probe experiments the excitation wavelength was either 400 nm or 520 nm. Probe pulses were generated in a non-collinear optical parametric amplifier, allowing for continuous wavelengths tunable over the visible spectrum from 470 nm to 700 nm.; The photolysis observed in 5′-deoxyadenosylcobalamin (coenzyme B₁₂) was independent of the excitation wavelength and resulted in the formation of a cob(II)alamin species and an adenosyl radical. Photolysis of the three other cobalamins was found to depend upon the excitation wavelength, generating an intermediate characteristic of a cob(III)alamin species.; 5′-deoxyadenosylcobalamin was studied in solvent mixtures of water and ethylene glycol. These studies showed that the intrinsic rate of recombination of adenosyl radical to the cobalamin was independent of the solvent mixture.; Finally, the photolysis measurements were extended to study the behavior of coenzyme B₁₂ bound to the protein glutamate mutase. Initial studies on glutamate mutase show that upon excitation at 400 nm the 5 ′-deoxyadenosylcobalamin forms a short lived intermediate state that is cob(III)alamin-like in nature. This intermediate is followed by bond homolysis to form the cob(II)alamin species observed for the free cofactor. Binding to the enzyme had only a small effect on the intrinsic rate of recombination. Free cobalamins have a recombination rate of circa 1.4 ns−1 while the recombination rate in the protein is 1.1 ns−1.