DNA photolyases are enzymes that use blue light to repair DNA that is damaged by UV light. Class I photolyases bind two cofactors - flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate polyglutamate (MTHF) – which are important for catalytic and light-harvesting activity, respectively. In our hypothesis, we propose that MTHF provides photoprotection to FAD from photodamage by photoinduced interactions between FAD and MTHF under continuous light-induced stress. Flavin molecules are known to form triplet states from which they can undergo electron transfer reactions that can potentially result in photodamage. The goals of our research include the determination of the possible interactions between MTHF and FAD in solution and the characterization of the FAD and MTHF triplet states at different timescales. To test our hypothesis, we have done long time-scale absorption experiments in solution to investigate whether there is an effect of MTHF on the rate of flavin degradation.  The results indicate that the presence of MTHF influences the rate of degradation of oxidized FAD at low concentrations.  Additional kinetic photodegradation studies with potassium bromide – a singlet excited state quencher – show that FAD stability is improved by the presence of Br<sup>-</sup>. Furthermore, purging the FAD solution to remove O<sub>2</sub>, a triplet state quencher, accelerates the FAD photodegradation.  Our current results support the hypothesis that MTHF may have a photoprotective role in solution and, possibly, in DNA photolyase and that the triplet states may play a role in the photoprotection of FAD by MTHF. Experimental approaches by using laser flash photolysis are currently undertaken to probe triplet state lifetimes of both FAD and MTHF, which can expand our understanding of possible triplet-triplet energy transfer and triplet state electron transfer reactions between these cofactors.

Spectroscopic investigation of photoinduced interactions between flavin adenine dinucleotide and 5,10-methenyltetrahydrofolate

ACS Spring 2022

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