1. Study of Proton Transfer in Escherichia coli Photolyase Meng Zhang Biophysics Graduate Program The Ohio State University June 21, 2013

2. Introduction Photolyase: Photoenzyme that utilizes blue light to repair UV- damaged DNA which causes skin cancer. Flavin cofactor: The catalytic cofactor of photolyase, flavin adenine dinucleotide (FAD), has five redox states.

3. UV-vis spectroscopic properties of wild type Photolyase The four redox states: FAD, FAD ⦁ ̶, FADH ⦁, and FADH ̶ have different UV-vis absorption spectra.

4. Redox conversions in EcPL mutants Site-directed mutagenesis N378C (N5 mutant) E363L N378C/E363L In the presence of substrate, N378C/E363L can stabilize at the FAD ̶ state under continuous white light for hours.  Proton channels are are eliminated completely.

5. The proposed proton channel in Photolyase The proton relay channel through E363 and surface water to N378 and then FAD; The proton diffusion channel through the substrate binding pocket.

6. Reaction kinetics k 12 k 21 k 32 k 23 k 43 k 34 WT N378C N378C with substrate

7. All in unit of min -1 k 12 k 21 k 32 k 23 k 43 k 34 k 12 k 21 k 23 k 32 k 34 k 43 k 43 with O 2 WT pH75.8×10 -4 0.150.313.3×10 -4 0.013<1.0×10 -4 0.025 pH90.330.714.1×10 -3 0.077<1.0×10 -4 8.2×10 -3 pH7 with S<1.0×10 -4 0.19<1.0×10 -4 2.5×10 -4 pH9 with S<1.0×10 -4 0.37<1.0×10 -4 5.6×10 -3 N378C pH76.7×10 -4 0.0880.050>10 <1.0×10 -3 0.023 pH9 0.0540.034>10 <1.0×10 -4 0.046 pH7 with S 8.3×10 -4 0.010>10<0.01 pH9 with S 2.2×10 -4 6.3×10 -4 >10<0.01 E363L pH79.7×10 -4 0.0150.018<1.0×10 -4 >1 pH9 0.0210.032<1.0×10 -4 >1 pH7 with S <1.0×10 -4 0.031<1.0×10 -4 0.012 N378C E363L pH7 0.0264.4×10 -3 >10 7.3×10 -3 Reaction kinetics

8. Proton transfer in (6-4) Photolyase and Cryptochromes (6-4) Photolyase Repairs (6-4) dipyrimidine photoproducts. Similar proton transfer pathway. Mutant N402C/E391L can stabilizes at FAD ̶ state in the presence of substrate. Cryptochromes (ongoing work) Insect Cryptochrome Plant Cryptochrome C-terminal domain FAD PHR domain

9. Conclusions N378 and E363 are the key residues for EcPL proton transfer. The proton channels in photolyase was proposed for the first time. Proton can be transported to flavin through two pathways: the proton relay channel (through E363 and N378 in EcPL); and the proton diffusion channel through the substrate binding pocket. Redox potentials of different FAD states are governed by the N5 residue and local environment. The proton transfer kinetics are critical to the mechanistic and functional divergence of Photolyase and Cryptochrome.

10. Acknowledgements Prof. Dongping Zhong Prof. Aziz Sancar (UNC) Ms. Lijuan Wang Dr. Jiang Li Zheyun Liu Chuang Tan And all other colleagues in the Zhong group! Funding from: NIH Thank you!