Due to the essential role of flavoproteins in light-driven biological activities, such as photoinduced DNA repair and signal transduction, it is of great interest to study the photochemistry and photophysics of flavin cofactor. This dissertation presents a systematic investigation on the ultrafast dynamics of flavin cofactor in four redox states in photolyase/cryptochrome family proteins. In photolyase, the antenna molecule MTHF absorbs a photon and transfer the excitation energy to the catalytic cofactor FADH¯ to enhance the DNA-repair efficiency. With the femtosecond-resolved fluorescence and transient absorption spectroscopy, we examined ultrafast dynamics of the resonance energy transfer from MTHF to three flavin redox states in photolyase and accurately determine the rates. The energy transfer from MTHF to the fully reduced hydroquinone FADH¯ occurs in 170 ps, but it takes 20 and 18 ps to the oxidized FAD and neutral semiquinone FADH•, respectively. The orientation factors were estimated from the acquired energy transfer rates and compared with values from the theoretical study. Both results demonstrate that MTHF-FADH¯ has the largest κ2 value, indicating the best orientation alignment for the physiologically functional state.
Photolyase uses blue light to repair the UV-induced pyrimidine dimer through a cylic electron transfer radical mechanism. The significant loss of repair efficiency by the mutation of the active-site residues indicates that those residues play a critical role in the DNA repair by photolyase. To understand how the protein active site modulates the repair and achieve the high efficiency, we mapped out the complete evolution of functional dynamics with 6 active-site mutated photolyases in real time, and analyzed the individual electron transfer processes in the catalytic reaction with Sumi-Marcus model. The results suggest that photolyase controls the critical electron transfer and the ring-splitting of pyrimidine dimer through modulation of the redox potentials and reorganization energies, and stabilization of the anionic intermediates, maintaining the dedicated balance of all the reaction steps and achieving the maximum function activity.
The flavin cofactor in photolyase is converted to oxidized FAD or neutral semiquinone FADH• and loses the enzyme activity. Upon light excitation, both FAD and FADH• can be photoreduced to catalytic form FADH¯ via electron transfer mainly through the neighboring conserved tryptophan triad. The ultrafast photoreduction dynamics of flavin cofactors in (6-4) photolyase was revealed by the femtosecond-resolved laser spectroscopy. Significantly, we found that the tryptophan triad has a reduction potential gradient modulated by the local protein environment, which promotes the electron hopping process with a distinctive directionality and enhances the photoreduction efficiency.
In Arabidopsis thaliana cryptochrome 2, we observed the ultrafast photoreduction of oxidized FAD in a few picoseconds and of neutral radical semiquinone FADH• in tens of picoseconds through intraprotein electron transfer. Such ultrafast dynamics exclude their potential roles as the functional states. In contrast, we found that the anionic hydroquinone FADH¯ and semiquinone FAD•- have complex deactivation dynamics on the time scale from a few picoseconds to a few nanoseconds, which is believed to occur through conical intersections with a flexible bending motion of the isoalloxazine ring. These results imply that the anionic hydroquinone FADH¯ is more likely to be the functional state rather than the two neutral states.