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An MD and QM/MM Study of the Active Site of (6-4) Photolyase

Although the crystal structure of (6-4) photolyase, isolated from D. melanogaster, has been recently solved, several mechanistic questions remain open. Two active-site histidines and a nearby tyrosine residue are thought to play crucial roles in catalysis. It was initially proposed that an oxetane intermediate was involved in the repair, but following the resolution of the crystal structure, Carell and coworkers suggested an alternative mechanism, in which one of the histidines is protonated.1 To test this assumption, and to determine a protonation state that deviates the least from the crystal structure, a set of 9 simulations with all possible combinations of protonation states of the active-site histidines were performed for three different oxidation states of FAD cofactor (FAD, FADH●, FADH-). As a complementary approach to molecular dynamics in finding the optimal protonation state, QM and QM/MM methods were employed to calculate the hyperfine couplings of certain hydrogen atoms in the FADH● radical, which was used as a probe of the local environment in an EPR/ENDOR study of the Xenopus laevis (6-4) photolyase.2 The results of these calculations and their comparison to the experimental values provide additional information concerning which protonation states of the histidine residues are most relevant in the enzyme.

(6-4) photolyase ; mechanism ; histidine protonation states ; EPR/ENDOR ; hyperfine coupling

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