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Redox Tuning of Flavin and Ultrafast Electron Transfer Mechanisms in DNA Repair by Photolyases

Zhang, Meng
http://rave.ohiolink.edu/etdc/view?acc_num=osu1469101235

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Biophysics.
The photolyase/cryptochrome superfamily is a vast and diversified class of flavoproteins that occurs in all kingdoms of life, utilizing the unique and versatile structure of flavin to catalyze difficult redox reactions in biological systems. The catalytic cofactor, flavin adenine dinucleotide (FAD), has four different redox states, and adopts a unique U-shape configuration in the active sites, with its lumiflavin and adenine moieties in close proximity. The redox tuning of photolyase/cryptochromes involve intra-protein electron transfer and proton transfer and plays an essential functional role. We systematically characterized the proton-transfer pathways in photolyase/cryptochromes, and with a double-site mutation that mimics the insect cryptochrome environment, we successfully eliminated the proton channels in photolyases for the first time. Proton is transported from protein surface to FAD through two pathways: the proton-relay pathway, and the active-site diffusion pathway. Kinetics of the redox conversions was solved and redox potentials of four states were determined. These results provide unprecedented insight into flavin redox tuning in each subfamily of photolyase/cryptochromes on the most fundamental level. Photolyase utilizes blue light to restore UV-induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), through a cyclic electron-transfer radical mechanism. Although the microbial class I photolyases have been characterized with great details in the past decades, studies on other classes lag behind. Here, using femtosecond spectroscopy we show our systematic dissection on various photolyases in all life branches. Complete repair cycles with ten elementary steps are solved for the first time for the single-stranded DNA photolyase from A. thaliana (AtCRY3), and the distantly related class II photolyases from D. melanogaster (DmPL), A. thaliana (AtPL), and M. mazei (MmPL). Strikingly, the critical electron-tunneling pathway for the class I photolyases lost dominance in these relatives. In AtCRY3, we observed a significantly slow direct electron tunneling occurring in 1560 ps, and the first-step hopping from lumiflavin to adenine occurs in 1800 ps, followed by the instantaneous second-step hopping from adenine to substrate in ~10 ps. The two-step hopping channel, which is negligible in class I photolyases due to slow intramolecular forward ET and ultrafast back ET, becomes competitive in AtCRY3. In class II photolyases, we observed similar electron bifurcation. Remarkably, in class II AtPL, the tunneling ET occurs extremely slowly in 6.5 ns, accompanying with a fast hopping ET in 565 ps, and the two-step hopping becomes dominant in repair. Therefore, a new, unified electron-transfer strategy for all photolyases with bifurcated routes through the conserved bent FAD cofactor is proposed. With structural differences in FAD and substrate binding, the time scales of the elementary steps involved in repair differ greatly throughout the highly diversified photolyase family. Nevertheless, in all photolyases, the initial electron injection manifests a bifurcation of direct electron tunneling and two-step electron hopping. Both pathways are operative in CPD repair with divergent efficiencies depending on distinct reduction potentials in different protein environment, and are converged at active site for efficient repair. From lower microbes to higher eukaryotes, the electron exploits from mainly direct tunneling along one route to dominant two-step hopping on the other path through evolution, to maintain decent repair efficiency and thus genome stability.
Dongping Zhong (Advisor)
Ralf Bundschuh (Committee Member)
Michael Poirier (Committee Member)
Zhengrong Wu (Committee Member)
144 p.
Biophysics
flavin; photolyase; DNA repair; electron transfer; ultrafast protein dynamics; femtosecond laser spectroscopy

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Citations

  • Zhang, M. (2016). Redox Tuning of Flavin and Ultrafast Electron Transfer Mechanisms in DNA Repair by Photolyases [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469101235

    APA Style (7th edition)

  • Zhang, Meng. Redox Tuning of Flavin and Ultrafast Electron Transfer Mechanisms in DNA Repair by Photolyases. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1469101235.

    MLA Style (8th edition)

  • Zhang, Meng. "Redox Tuning of Flavin and Ultrafast Electron Transfer Mechanisms in DNA Repair by Photolyases." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469101235

    Chicago Manual of Style (17th edition)

osu1469101235
489
© 2016, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.