Spectroscopic exploration of fundamental electronic parameters of flavins in an effort to elucidate photoinduced electron transfer in DNA photolyase

Flavins function as coenzymes in many biological reactions. Studies have employed optical spectroscopy of flavins and flavoproteins. The interpretation of these optical spectroscopic studies requires knowledge of the fundamental electronic parameters that influence the interaction between light and flavins. This is very significant during light-dependent processes, such as photoinduced electron transfer in DNA photolyase. Despite this there is little experimental information about such fundamental electronic parameters.;The initial electron acceptor of photoinduced electron transfer in DNA photolyase has not been determined. Fluorescence Stark spectroscopy can determine the initial electron acceptor. The analysis of such studies requires very-high-quality low-temperature absorption spectra and the electronic transition dipole moment directions. To obtain low-temperature absorption spectra, a cryogenic optical waveguide spectrometer (COWS) was developed and used to perform the studies in Chapter 3. COWS uses microliter samples and provides significant improvements in sensitivity. COWS demonstrated a high reproducibility and detected 15 ng of flavoprotein. Using LD spectroscopy, the electronic transition dipole moments of reduced flavin were determined. TD-DFT was also used to calculate the normal modes and the electronic transition dipole moments of reduced flavin. The directions of the electronic transition dipole moments in the 200-450 nm region are: 79°+/-4° at 350 nm and 93°+/-4° at 290 nm counterclockwise to the N5-N10 axis. Stark spectroscopic studies on oxidized and reduced flavins were performed. For the oxidized flavin, the change in dipole moment for the S0→S1 transition is about 3 times smaller than that for the S0→S2 transition. The direction of the S1 dipole was determined to lie roughly parallel with the ground-state dipole moment. The results indicated that the S 2 dipole moment points toward N10. The polarizability change for these two transitions suggests that the S0 and S1 states have about the same polarizability, while the S2 state is significantly more polarizable than the lower energy states.