Ultrafast dynamics of flavin cofactor in DNA repair by photolyase and in signaling formation of cryptochrome

Due to the essential role of flavoproteins in light-driven biological activities, such as photoinduced DNA repair and signal transduction, photochemistry and photophysics of flavins have drawn considerable attention. This dissertation presents a systematic investigation on the ultrafast dynamics of flavin in five redox states in solution and in flavoproteins, especially in CPD photolyase and insect Type 1 cryptochrome. With highly sensitive femtosecond-resolved transient absorption and fluorescence spectroscopy techniques, we followed the weak absorption and fluorescence of flavin-related transient species, respectively, in photoinduced processes. Significantly, we observed a strong correlation between their excited-state dynamics and the planarity of their flavin isoalloxazine ring. For a bent ring structure, we all observed ultrafast dynamics from a few to hundreds of picoseconds and strong excitation-wavelength dependence of emission spectra, indicating deactivation during relaxation. A butterfly bending motion is invoked to get access to conical intersection(s) to facilitate deactivation. These states include the anionic radical semiquinone in proteins and fully-reduced neutral and anionic hydroquinones in solution. In a planar configuration, flavins have a long lifetime in nanoseconds except for the stacked conformation of oxidized flavin, where the intramolecular electron transfer between the ring and the adenine moiety in 5-9 ps as well as the subsequent charge recombination in 30-40 ps were observed.;In photolyases and cryptochromes, we observed ultrafast photoreduction of oxidized state in subpicosecond and of neutral radical semiquinone in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that the anionic semiquinone and hydroquinone have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the anionic hydroquinone is the excited active state and has a long (nanosecond) lifetime optimal for DNA-repair function. In insect Type1 cryptochromes known to be blue-light photoreceptors the excited active form is suggested to be the anionic semiquinone and has complex deactivation dynamics on the time scales from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.;Upon function initiation by blue light, we observed a continuous active-site relaxation which strongly couples with catalytic electron transfer reactions and the direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site with no net electron change in redox states of the flavin cofactor. To achieve high repair efficiency, the efficiency of both charge-separation and ring-splitting reactions must be optimized to minimize the nonproductive pathway of charge recombination before ring cleavage. These observed synergistic motions in the active site of the damaged DNA-enzyme complex reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.