Charge Redistribution in Oxidized and Semiquinone E. coli DNA Photolyase upon Photoexcitation: Stark Spectroscopy Reveals a Rationale for the Position of Trp382 Goutham Kodali, † Salim U. Siddiqui, ‡ and Robert J. Stanley* ,† Department of Chemistry, Temple UniVersity, Philadelphia, PennsylVania 19122, and Department of Radiation Oncology, Henry Ford Hospital, Detroit, Michigan 48202 Received November 25, 2008; E-mail: rstanley@temple.edu Abstract: The electronic structure of the two lowest excited electronic states of FAD and FADH • in folate- depleted E. coli DNA photolyase (PL OX and PL SQ , respectively) was measured using absorption Stark spectroscopy. The experimental analysis was supported by TDDFT calculations of both the charge redistribution and the difference dipole moments for the transitions of both oxidation states using lumiflavin as a model. The difference dipole moments and polarizabilities for PL OX are similar to those obtained in our previous work for flavins in simple solvents and in an FMN-containing flavoprotein. No such comparison can be made for PL SQ , as we believe this to be the first experimental report of the direction and magnitude of excited-state charge redistribution in any flavosemiquinone. The picture that emerges from these studies is discussed in the context of electron transfer in photolyase, particularly for the semiquinone photoreduction process, which involves nearby tryptophan residues as electron donors. The direction of charge displacement derived from an analysis of the Stark spectra rationalizes the positioning of the critical Trp382 residue relative to the flavin for efficient vectorial electron transfer leading to photoreduction. The ramifications of vectorial charge redistribution are discussed in the context of the wider class of flavoprotein blue light photoreceptors. Introduction DNA photolyase is a light-driven flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) in UV-damaged DNA. 1,2 The protein is found in all kingdoms, including nonplacental mammals. Two noncovalently bound cofactors are associated with PL. One is an absolutely conserved flavin adenine dinucleotide (FAD) molecule without which the protein cannot bind or repair substrate. The second cofactor appears to function as a photoantenna, although the protein can bind and repair DNA without it. Photolyase binds to the CPD in a light-independent manner. Substrate binding does not depend to any significant degree on the oxidation state of the flavin. 3,4 The earliest crystal structure of photolyase revealed a surface-accessible cavity with the approximate dimensions of a CPD, and it was suggested that base flipping of the CPD was required to bring the substrate close to the flavin cofactor found at the base of this cavity. 5 This base flipping hypothesis, tested by several indirect means, 6-8 was ultimately confirmed by a crystal structure of PL with a modified substrate molecule. 9 The FAD must be fully reduced (FADH - , PL RED ) for repair to take place, and we 10 and others 11-14 have shown that direct excitation of the FADH - cofactor leads to efficient CPD repair through ultrafast electron transfer from *FADH - to an as yet unknown initial acceptor. Ultimately, the radical anion of the dimer, CPD •- , is formed, although definitive evidence for this intermediate is still lacking, and the flavin becomes oxidized to FADH • . FADH • is therefore an intermediate in the electron transfer pathway. At some point in the reaction cycle, the CPD •- undergoes a thermally activated monomerization reaction 15,16 to yield a thymine and thymine radical anion, T -• . Back electron transfer from FADH • -T -• fFADH - -T brings the flavin back † Temple University. ‡ Henry Ford Hospital. (1) Kay, C. W. M.; Bacher, A.; Fischer, M.; Richter, G.; Schleicher, E.; Weber, S. Compr. Ser. Photochem. Photobiol. Sci. 2006, 6, 151– 182. (2) Sancar, A. Chem. ReV. 2003, 103, 2203–2237. (3) Sancar, G. B.; Jorns, M. S.; Payne, G.; Fluke, D. J.; Rupert, C. S.; Sancar, A., III. J. Biol. Chem. 1987, 262, 492–8. (4) Jorns, M. S.; Sancar, G. B.; Sancar, A. Biochemistry 1985, 24, 1856– 61. (5) Park, H.-W.; Kim, S.-T.; Sancar, A.; Deisenhofer, J. Science 1995, 268, 1866–72. (6) Berg, B. J. V.; Sancar, G. B. J. Biol. Chem. 1998, 273, 20276–20284. (7) Butenandt, J.; Burgdorf, L. T.; Carell, T. Angew. Chem., Int. Ed. 1999, 38, 708–711. 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Published on Web 03/17/2009 10.1021/ja809214r CCC: $40.75  2009 American Chemical Society J. AM. CHEM. SOC. 2009, 131, 4795–4807 9 4795