Stanley, Robert J.; Valentine, Ann M.; Voelz, Vincent; Grubmeyer, Charles (Temple University. Libraries, 2016)
      ABSTRACT Many enzymes require cofactors in order to carry out specific functions. Flavins, which are naturally fluorescent, compose a unique group of redox cofactors because they have the ability to transfer one or two electrons and are therefore found in three different oxidation states. A specific flavin, flavin adenine dinucleotide (FAD), is a crucial cofactor that facilitates electron transfer in many flavoproteins involved in DNA repair, photosynthesis, and regulatory pathways. One example of a FAD-containing DNA repair protein is DNA Photolyase (PL). E. coli PL is a monomeric flavoprotein that facilitates DNA repair via a photoinduced electron transfer reaction. The catalytic cofactor, FAD, transfers an electron to a thymidine dimer lesion, to cleave the cyclobutane ring and restore the DNA strand. Although the mechanism of repair has been partially elucidated by our group, it is still unclear whether or not the electron is transferred directly from the isoalloxazine moiety to the dimer or if the electron hops from the isoalloxazine moiety to the adenine moiety to the dimer. This sequential hopping mechanism should have excited state absorption features for the reduced flavin species, an adenine radical anion, and the semiquinone flavin species. To investigate the mechanistic role of adenine, E. coli PL has been reconstituted with -FAD, an FAD analogue in which the adenine was substituted via chemical means with 1,N6 – ethenoadenine dinucleotide. -FAD was selected due to its ease of synthesis and because its structure changes the thermodynamic driving force for the electron transfer reaction, by lowering the energetic gap (LUMO-LUMO) between the isoalloxazine ring and the modified adenine. In order to characterize the excited state dynamics of the mutant chromophore, the transient absorption measurements were made of each free flavin in solution. These measurements indicate the pathway of electron transfer must be mediated via superexchange rather than a hopping mechanism. This important result shows that the role of adenine in photolyase is to facilitate a superexchange electron transfer mechanism, and a modified flavin can act as a reporter under these experimental conditions. By exploiting Corynebacterium ammoniagenes FAD synthetase adenylation promiscuity, we have enzymatically-synthesized and purified a novel dually fluorescent flavin cofactor. This new flavin adenine dinucleotide (FAD) analogue, flavin 2-aminopurine (2Ap) dinucleotide (F2ApD), can be selectively excited through the 2Ap moiety at 310 nm, a wavelength at which flavins have intrinsically low extinction. The dinucleotide 2Ap emits at 370 nm with high efficiency. This emission has excellent overlap with the absorption spectra of both oxidized and reduced hydroquinone flavin (FlOX and FlHQ respectively), which emit at ~525 and ~505 nm respectively. We have characterized the optical properties of this dually fluorescent flavin, iFAD. Steady state fluorescence excitation and emission spectra were obtained and contrasted with the other flavins. Temperature- and solvent-dependent emission spectra suggest that F2ApD stacking interactions are significantly different compared to FAD and etheno-FAD (FAD). The optical absorption spectra of these dinucleotides were compared with FMN to explore electronic interactions between the flavin and nucleobase moieties. To probe the evolution of the different excited state populations, femtosecond transient absorption measurements were made on the iFADs, revealing that F2ApD exhibited unique transient spectra as compared to either FAD or FAD. The significance of these results to flavins, flavoprotein function, and bioimaging are discussed. The reconstituted -FAD in E.coli photolyase was catalytically active and actually repaired more efficiently than the FAD-reconstituted photolyase. To validate that an enzymatically synthesized iFAD could be reconstituted into a flavoprotein, this work shows a DNA repair assay using F2ApD that was reconstituted into E. coli photolyase, generating the reconstituted analogue, ApPL. Activity assays were compared between FAD-PL and ApPL. This comparison further elucidates the importance of the driving force on the electron transfer reaction in PL. A comparison of fluorescence spectroscopies between the reconstituted PLs highlights their applicability as biosensors and/or mechanistic reporters.