Study of Proton Transfer in Escherichia coli PhotolyaseMeng Zhang

Biophysics Graduate Program

The Ohio State University

June 21, 2013

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.

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

UV-vis absorption spectra.

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.

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.

Reaction kineticsFADox FAD•- FADH⦁ FADH-

k12

k21 k32

k23

k43

k34

WT

WTN378C

N378C with substrate

 

 

 

 

All in unit of min-1

FADox FAD•- FADH⦁ FADH-k12

k21 k32

k23

k43

k34

k12 k21 k23 k32 k34 k43 k43 with O2

WT

pH7 5.8×10-4 0.15 0.31 3.3×10-4 0.013 <1.0×10-4 0.025

pH9 0.33 0.71 4.1×10-3 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

pH7 6.7×10-4 0.088 0.050 >10 >10 <1.0×10-3 0.023

pH9   0.054 0.034 >10 >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 

pH7 9.7×10-4 0.015 0.018 <1.0×10-4 >1    

pH9   0.021 0.032 <1.0×10-4 >1    

pH7 with S   <1.0×10-4 0.031 <1.0×10-4 0.012    

N378C E363L

pH7   0.026 4.4×10-3 >10 >10 7.3×10-3  

Reaction kinetics

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

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.

Acknowledgements

Prof. Dongping ZhongProf. Aziz Sancar (UNC)

Ms. Lijuan WangDr. Jiang LiZheyun LiuChuang Tan

And all other colleagues in the Zhong group!

Funding from: NIH

Thank you!