An overexposure to ultraviolet radiation can cause sunburn and some forms of skin cancer. UV light causes many different photoproducts. The cys-syn cyclobutylpyrimidine dimer (CPD) is the major photoproduct upon UV irradiation. DNA photolyase (PL) is a light-driven flavoprotein that repairs CPD in UV-damaged DNA. This repair process occurs in the presence of blue light through ultrafast photo-induced electron transfer from reduced anionic flavin adenine dinucleotide (FADH¯) to the CPD by an unknown mechanism. Since the excited state flavin transfers an electron to repair the damaged DNA, it is of utmost importance that we understand better the excited state properties of the flavins. In this work the excited state electronic properties of all three-oxidation states of flavin: oxidized form (FAD), semiquinone radical form (FADH•) and reduced anionic form (FADH¯) were studied using Stark spectroscopy and complimented by time dependent density functional theory (TD-DFT) calculations. These results are presented and discussed in Chapter 3 and 4. The difference dipole moments (Δμ) and the difference polarizabilities (Tr(Δα01)) were experimentally determined for first two lowest optically accessible states. The results are discussed in the context of photoreduction of flavins in wider class of flavoprotein blue light photoreceptors and catalytic electron transfer process in DNA repair. In the later part of this thesis (Chapters 5 and 6) the excited state electronic properties of monomeric 2-Aminopurine (2AP), 8-Vinyladenine 8VA were presented. These 8VA, 2AP are examples of fluorescent nucleotide analogues of adenine that can be incorporated into DNA with little perturbation of the normal double-helical structure. The fluorescence of these analogues is quenched when incorporated in double-stranded DNA (dsDNA). The basic mechanism underlying the fluorescence quenching by base stacking of 2AP and 8VA are is not well understood, and thus exploring the excited state electronic structures of these bases is an important first step. We have explored the excited state properties of 2AP and 8VA in frozen LiCl and ethanol solutions using Stark spectroscopy. High-level ab initio and TD-DFT calculations were performed to compliment the experimental results. / Chemistry

The excited state electronic structures of several biologically important chromophores were studied by Stark spectroscopy. The extent of charge redistribution upon excitation to the lowest excited states of the oxidized and semiquinone forms of flavin adenine dinucleotide (FAD) bound to the light activated DNA repair enzyme DNA photolyase have been studied previously by this technique. This work focuses on the catalytically active form, the two-electron reduced anion. To facilitate analysis of this experiment, the Stark spectra of a simple flavin derivative that is soluble in organic solvents was measured. The results of the analysis of these data are in agreement with previously a published linear dichroism experiment that found the absorption spectrum of flavins in this redox state arises from two distinct electonic transitions in the visible/near-ultraviolet spectral range, a fact that has not been incorporated into the analysis of many ultrafast spectroscopic experiments of reduced anionic flavins/flavoproteins. The difference dipole moment of the second, more intense, transition was found to be about twice as large as that of the lowest energy transition. With the aid of ab initio calculations, the directions of these dipole moments in the molecular frame were assigned. For both transitions, it was found that negative charge density is shifted toward the xylene ring of the flavin upon excitation. Another important consideration for the correct analysis of the photolyase spectra is the possibility of contamination by small amounts of the antenna chromophore, which also has absorption intensity in the near-ultraviolet region. We chose to study the cofactor for E. coli photolyase, 5,10-methenyltetrahydrofolate, and its photodecomposition product, 5,10-methylenetetrahydrofolate. The difference dipole moments for the lowest energy transitions of both of these chromophores were found to be quite large, ranging from 9-12 D fc and lying primarily along the transition dipole moment. Additionally, the difference polarizability of both chromophores was large, on the order of 200-300 Å3 fc2 . The Stark spectra of reduced anionic FAD in photolyase agrees well with the findings of the experiments on flavin in organic solvent; the magnitude of the difference dipole moments in both cases match within experimental error. While the direction of the difference dipole moment for the lowest transition is also the same in both cases, that of the second transition is changed in the protein matrix. The assignment of these vectors in the molecular frame shows that the two dipole moments are coincident for the cofactor bound to photolyase. This finding, where electron density is shifted toward the point of the flavin ring closes to the DNA lesion bound to the enzyme, is strong evidence that direct electron transfer takes place from the isoalloxazine ring of FAD to the DNA substrate in the catalytic cycle. The usefulness of Stark spectroscopy in investigating photoinduced charge redistribution was also shown for the donor-π-acceptor flavin dyad, azobenzylflavin (ABFL). The difference dipole moment was found to be 22 D, an approximately three-fold increase from the largest difference dipole moment found in naturally occurring flavins. This extensive charge redistribution corresponds to a large hyperpolarizability of the chromophore that suggests that ABFL may be useful in nonlinear optical applications. Transient absorption was used to supplement these experiments by monitoring the decay kinetics of ABFL after excitation. It was found that ABFL undergoes ultrafast charge recombination within 6 ps after excitation, leading to depopulation of the charge separated state before useful work can be performed for applications requiring electron transfer. These studies provide the ground work for rational design of other ABFL-like derivatives for use in a variety of applications. / Chemistry

Photoinduced electron transfer (PET) plays a crucial role in a wide array of biological pathways. These electron transfer reactions happen from or to the excited state of a chromophore upon absorption of light. Hence understanding the properties of excited states is necessary in elucidating the details of such pathways. The work presented in this thesis deals with PET in two systems: Fluorescent Nucleobase Analogues (FBAs) and DNA photolyase. The introductory chapter (Chapter 1) presents some background information about the two systems and sets up the stage for the reasoning behind the problems addressed in this thesis. FBAs are fluorescent analogues of naturally occurring, weakly fluorescent native nucleic acid bases. When incorporated into single stranded (ss) or double stranded (ds) DNA, the FBA fluorescence is significantly quenched. PET has been implicated to be the cause for the observed quenching. Here we have presented our attempt to correlate the quenching behavior of free FBA: nucleic acid monophosphate (NMP) pairs with the free energies associated with excited state electron transfer delta GET. Based on the delta GET values, we have tried to assign the direction of electron transfer. The quenching behavior of the FBA:NMP pairs were studied through Stern-Volmer (SV) quenching and time-resolved fluorescence studies. The above described analysis has been applied on FBAs: 4-amino-6-methyl-8-(2'-deoxy-beta-D-ribofuranosyl)-7(8H)-pteridone (6MAP), 4-amino - 2, 6 - dimethyl - 8 - (2'-deoxy-beta-d-ribofuranosyl) -7(8H) - pteridone (DMAP), 3-methyl-8-(2'-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3MI) and 6-Methyl-8-(2'-deoxy-β-D-ribofuranosyl) isoxanthopterin (6MI) (Chapter 3), 2-Aminopurine (2AP) (Chapter 4), 8-Vinyl Adenosine (8VA) (Chapter 5). The final part of this thesis (Chapter 6) is on understanding the mechanistic details of a DNA repair process that is due to photoinduced electron transfer in DNA photolyase, a flavoprotein. Before the electron reaches the damaged site in the DNA, the initial electron acceptor in this repair process has been speculated to be the adenine of the flavin adenine dinucleotide (FAD). We have tested this hypothesis by measuring and comparing the various kinetic parameters associated with this process by reconstituting into apo-photolyase the natural cofactor of photolyase (FAD) and an adenine modified flavin (Etheno FAD, epsilon FAD). / Chemistry

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