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Ultrafast Dynamics of Intramolecular Electron Transfer and DNA Repair by Photolyase

Liu, Zheyun
http://rave.ohiolink.edu/etdc/view?acc_num=osu1373534794

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Chemistry.
One of the detrimental effects of ultraviolet (UV) radiation on DNA is the formation of the cyclobutane pyrimidine dimer (CPD) and a less frequently occurring (6-4) photoproduct (6-4PP) between two adjacent pyrimidine rings. These UV-induced photolesions interfere with replication and transcription, and may result in mutation and cell death. Photolyase, a class of flavoprotein, uses blue light to restore the CPD and 6-4PP to two normal bases. The cofactor, flavin adenine dinucleotide (FAD), has an unusual bent configuration in photolyase and cryptochrome and such a folded structure may have a functional role in initial photochemistry. We report here our systematic characterization of cyclic intramolecular electron-transfer (ET) dynamics between the flavin and adenine moieties in four redox forms of the oxidized, neutral and anionic semiquinone, and anionic hydroquinone states. These four ET dynamics unambiguously determine only the anionic hydroquinone flavin as the functional state in photolyase due to the slower ET dynamics with the adenine moiety and a faster ET dynamics with the substrate, while the intervening adenine moiety mediates such electron tunneling for repair of damaged DNA. Utilizing ET as a universal mechanism for photolyase and cryptochrome, these results support an anionic flavin as the active state for the blue-light photoreceptor cryptochrome. With the understanding that the anionic hydroquinoid flavin is the functional redox state in photolyase, we further studied the dynamics of damaged DNA repair by photolyase. Based on the previously resolved radical mechanism, two fundamental issues, the mechanism of cyclobutane ring splitting and the pathway of electron tunneling, were ready to be resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance dynamics of all catalytic reactions. Besides that, we found that the adenine moiety of the unusual bent cofactor is essential to mediating all electron transfer dynamics through a super-exchange mechanism. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine enable this complex photorepair machinery to achieve the maximum repair efficiency close to unity. Finally, we revealed the mechanism of 6-4PP restoration by photolyase for the first time. Using site-directed mutagenesis and femtosecond-resolved absorption spectroscopy, we determined that the key step in the repair photocycle is a cyclic proton transfer between the enzyme and the substrate and leading to 6-4PP repair in tens of nanoseconds. These key dynamics define the repair photocycle and explain the underlying molecular mechanism of the enzyme's modest efficiency.
Dongping Zhong (Advisor)
Heather Allen (Committee Member)
Karin Musier-Forsyth (Committee Member)
Sherwin Singer (Committee Member)
131 p.
Chemistry
Ultrafast enzyme dynamics; Protein electron transfer; Laser spectroscopy

Recommended Citations

Citations

  • Liu, Z. (2013). Ultrafast Dynamics of Intramolecular Electron Transfer and DNA Repair by Photolyase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373534794

    APA Style (7th edition)

  • Liu, Zheyun. Ultrafast Dynamics of Intramolecular Electron Transfer and DNA Repair by Photolyase . 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1373534794.

    MLA Style (8th edition)

  • Liu, Zheyun. "Ultrafast Dynamics of Intramolecular Electron Transfer and DNA Repair by Photolyase ." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1373534794

    Chicago Manual of Style (17th edition)

osu1373534794
815
© 2013, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.