State-of-the-art femtosecond to nanosecond time-resolved spectroscopic techniques are being used to study reaction mechanism in complicated biological systems. Current research is focused on investigating the reaction mechanism in B12 dependent enzymes.

The key catalytic element in B12 is a unique carbon - cobalt bond. Reactivity in B12 enzymes involves homolysis (adenosylcobalamin dependent enzymes) or heterolysis (methylcobalamin dependent enzymes) of the active carbon-cobalt bond. Although the B12 catalysis is not naturally photoinitiated, a photon can be used to trigger bond homolysis in both enzyme bound and free B12 coenzymes.

In a second project, related to our studies of cyclohexadiene, femtosecond time-resolved deep ultraviolet spectroscopy is being used to investigate polyene photochemistry. Vitamin D3 is produced through ultraviolet photolysis of 7-dehydrocholesterol in the skin. The initial step is the ring-opening reaction of 7-dehydrocholesterol to form pre-vitamin D3. In our laboratory we have been investigating the photochemical ring opening reaction of both 1,3-cyclohexadiene and the more complicated 7-dehydrocholesterol chromophore.

The S1 excited state of cobalamins is characterized by a spectrum blue-shifted with respect to the ground state spectrum (Type I) or a spectrum resembling the ground state spectrum (Type II). These spectra are illustrated as the blue lines in the plot to the right.

Transient absorption measurements of adenosylcobalamin bound to glutamate mutase highlight the importance of the electrostatic environment on the coenzyme. Coenzyme B12 has a type I excited state in solution - either water of ethylene glycol. Binding to the protein modifies the nature of the excited state - to a type II state. The protein also slows down the intrinsic rate for geminate recombination of the radicals. These results suggest that direct interactions between the protein and the adenosyl group may also play an important role in activating the C-Co bond.

This research is supported by the National Science Foundation. Some early work on cobalamins was supported by the NIH.