ULTRAFAST DYNAMICS OF ENERGY AND ELECTRON TRANSFER IN DNA-PHOTOLYASE DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Chaitanya Saxena, M.Phil. ****** The Ohio State University 2007 Dissertation Committee: Professor Dongping Zhong, Advisor Approved by Professor Richard P. Swenson Professor Zucai Suo Professor Michael G. Poirier Advisor Biophysics Graduate Studies Program ABSTRACT One of the detrimental effects of UV radiation on the biosphere is the formation of cyclobutane pyrimidine dimers (Pyr<>Pyr) between two adjacent thymine bases in DNA. Pyr<>Pyr dimers can not be repaired by normal DNA repair machinery and may result in gene mutation or cell death. Photolyase, a photoenzyme harnesses blue or near- UV light energy to cleave the cyclobutane ring of the Pyr<>Pyr and thus protects against the harmful effects of UV radiation. In the proposed hypothesis for the catalysis, the enzyme binds a Pyr<>Pyr in DNA, independent of light. The photoantenna, a photolyase cofactor methenyltetrahydrofolate (MTHF) harvests a UV/blue-light photon, and transfers the excitation energy (dipole-dipole interaction) to another photolyase cofactor, a fully reduced flavin (FADH−). Excited FADH−* then transfers an electron to the Pyr<>Pyr, which consequently splits the Pyr<>Pyr into two pyrimidine moieties and hence repairs the damaged DNA. As proposed, the repair cycle ends when the excess electron from the repaired pyrimidine moieties is transferred back to the nascent-formed neutral FADH• species and regenerates the active FADH− form. The complex mechanism of energy and electron transfer in photolyase enzyme involved in performing its DNA repair function was investigated using femtosecond-resolved fluorescence up- conversion and transient absorption methods. Under physiological conditions, the excitation energy transfer from the antenna molecule MTHF to the FADH− occurs in 292 ii ps, but it takes 19 ps to the in vitro oxidized neutral cofactor FADH•. The orientation factors were found to be 0.11 for the MTHF- FADH− pair and 0.28 for MTHF- FADH•, unfavorable for energy transfer, indicating the existing structural constraints probably placed by three functional binding sites. The photoreduction of the neutral FADH• to the catalytically active cofactor FADH− was revealed to evolve along two electron-transfer pathways: one is along a tryptophan triad with the initial electron hop in 10 ps; the other route starts with an initial electron separation in 40 ps through the neighboring phenylalanine followed by either tunneling along an α-helix or hopping through the tryptophan triad again. Ultrafast libration/rotation motions of local protein residues and trapped water molecules at the active site were observed to initially occur in ~2 ps. These ultrafast ordered-water motions are critical to stabilizing the photoreduction product FADH− instantaneously to prevent fast charge recombination. Monitoring the catalytic processes we observed direct electron transfer from the FADH−* to the Pyr<>Pyr in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site, with no net change in the redox state of the flavin cofactor. The photophysics of FADH− cofactor was studied in aqueous solution. Dramatic shortening of the excited state lifetime of FADH− in aqueous solution compare to its iii lifetime in protein environment compelled us to propose that enzyme photolyase also modulates photophysical properties of the flavin cofactor to perform the essential biological function of electron transfer to repair damaged DNA. iv Dedicated to Family, Friends, And Gurus v ACKNOWLEDGMENTS I would like to thank my advisor, Prof. Dongping Zhong, for his unwavering support and guidance throughout my graduate education. His guidance and insight were instrumental in the success of the research presented in this dissertation. I am grateful to Prof. Aziz Sancar, University of North Carolina (Chapel Hill) who conceded his contagious motivation for pursuing DNA-repair research and shared more than two decades of research experience in planning the key experiments. I am thankful to Lijuan Wang and Ya-Ting Kao who closely remain involved in the present work and provided help and insight in performing and analyzing the experiments. I would like to thank Prof. Richard P. Swenson and Prof. Russ Hill for helping me in developing understanding of flavin biochemistry and photochemistry. I would like to extend my thanks to Prof. Dehua Pei, of the Ohio State University who willingly shared his resources for our bacterial growth needs during initial time of this work. I am also thankful to Kari Green-Church and Chunhua Yuan of the Ohio State University Campus Chemical Instrument Center (CCIC) for the technical support that they provided me for the characterization of photolyase substrates using Mass-spectrometry and Nuclear vi Magnetic Resonance techniques. Thanks are also due for Prof. Michael G. Poirier who shared his resources for purification of the photolyase substrates. The Ohio State Biophysics Graduate Studies Program and The Department of Physics, The Ohio State University deserves special thanks for allowing me to use all the available administrative and technical facilities to perform this work and advance my research career. I would like to thank all the members of Zhong group for their stimulating discussions and for their help throughout the research. Very special thanks to my wife Dr. Yanping Yan who provided moral support, encouragement and professional help throughout my doctoral work. This research was supported by the funds available to Prof. Dongping Zhong from ‘Department of Physics’ and ‘National Institute of Health’ and to research collaborator Prof. Aziz Sancar from ‘National Institute of Health’. Partial support to this research also comes from ‘American Heart Association’ in the form of Predoctoral fellowship. vii VITA August 28, 1975, _______________________________________________________ Born-Dewas, India 1993-1996 _________________________ B.Sc. Chemistry (honors), Devi Ahilya University, Indore India 1996-1998 ___________________ M.Sc. Pharmaceutical Chemistry, Devi Ahilya University, Indore India 1998-1999 _______________________ ‘Production Chemist’, Lupin Laboratories Pvt. Ltd. Bhopal, India 1999-2001 ___________________________________ M.Phil. Biophysics, NIMHANS, Bangalore , India 2001-present________________ Graduate Research Associate, The Ohio State Universty, Coumbus, USA PUBLICATIONS C. Saxena, Y.-T. Kao, L. Wang, A. Sancar and D. Zhong “Direct observation of DNA repair by photolyase” in Femtochemistry VII: Fundamental Ultrafast Processes in Chemistry, Physics, and Biology, A. W. Castleman, Jr., M. L. Kimble, eds., Elsevier: Amsterdam (2006) p407. Y.-T. Kao*, C. Saxena*, L. Wang, A. Sancar and D. Zhong “Direct observation of thymine dimer repair in DNA by photolyase” Proc. Natl. Acad. Sci. USA 102:16128- 16132 (2005). (*authors contributed equally to this work) viii C. Saxena, H. Wang, I. H. Kavakli, A. Sancar and D. Zhong “Ultrafast dynamics of resonance energy transfer in cryptochrome” J. Am. Chem. Soc. 127:7984-7985 (2005). H. Wang, C. Saxena, D. Quan, A. Sancar and D. Zhong “Femtosecond dynamics of flavin cofactor in DNA photolyase: Radical reduction, local solvation, and charge recombination” J. Phys. Chem. B. 109:1329-1333 (2005). C. Saxena, A. Sancar and D. Zhong “Femtosecond dynamics of DNA photolyase: Energy transfer of antenna initiation and electron transfer of cofactor reduction” J. Phys. Chem. B 108:18026-18033 (2004). FIELDS OF STUDY Major Field: Biophysics ix TABLE OF CONTENTS Page Abstract ……………………………………………………………………………………………………...ii Dedications ………………………………………………………………………………………………….v Acknowledgments …………………………………………………………………………………………..vi Vita...……………………………………………………………………………………………………….viii List of Tables ..……………………………………………………………………………………………...xv List of Figures……………………………………………………………………………………………...xvi Abbreviation..………………………………………………………………………………………………xix Chapter 1 Introduction....................................................................................................................... 1 1.1 Enzyme dynamics............................................................................................ 1 1.1.1 Enzyme catalysis and enzyme dynamics ................................................. 2 1.1.2 Dynamic fluctuations in enzyme ............................................................. 3 1.1.3 Time-resolved approaches to characterize enzyme dynamics ................. 5 1.1.4 Applications ........................................................................................... 10 1.2 DNA photolyase............................................................................................. 11 1.2.1 Biological function................................................................................. 11 1.2.2 Significance............................................................................................ 12 x 1.2.3 The photolyase enzyme.......................................................................... 13 1.2.4 The damaged DNA substrate................................................................. 17 1.2.5 Molecular mechanism