1. Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase.
- Author
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Schleicher E, Hitomi K, Kay CWM, Getzoff ED, Todo T, and Weber S
- Subjects
- Animals, DNA Damage radiation effects, Deoxyribodipyrimidine Photo-Lyase genetics, Deoxyribodipyrimidine Photo-Lyase metabolism, Electron Spin Resonance Spectroscopy, Flavin-Adenine Dinucleotide genetics, Flavin-Adenine Dinucleotide metabolism, Histidine genetics, Histidine metabolism, Hydrogen-Ion Concentration, Kinetics, Models, Chemical, Point Mutation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Structure-Activity Relationship, Ultraviolet Rays, Xenopus laevis, DNA Repair radiation effects, Deoxyribodipyrimidine Photo-Lyase chemistry, Ethers, Cyclic chemistry, Flavin-Adenine Dinucleotide chemistry, Histidine chemistry
- Abstract
(6-4) photolyase catalyzes the light-dependent repair of UV-damaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH., which is believed to be transiently formed during the enzymatic DNA repair. Here (6-4) photolyase has been examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. Characterization of selected proton hyperfine couplings of FADH., namely those of H(8alpha) and H(1'), yields information on the micropolarity at the site where the DNA substrate is expected to bind. Shifts in the hyperfine couplings as a function of structural modifications induced by point mutations and pH changes distinguish the protonation states of two highly conserved histidines, His(354) and His(358), in Xenopus laevis (6-4) photolyase. These are proposed to catalyze formation of the oxetane intermediate that precedes light-initiated DNA repair. The results show that at pH 9.5, where the enzymatic repair activity is highest, His(358) is deprotonated, whereas His(354) is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct.
- Published
- 2007
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