Photolyases (PHRs) are DNA repair enzymes that revert UV-induced photoproducts, either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts (PPs), into normal bases to maintain genetic integrity. (6-4) PHR must catalyze not only covalent bond cleavage, but also hydroxyl or amino group transfer, yielding a more complex mechanism than that postulated for CPD PHR. Previous mutation analysis revealed the importance of two histidines in the active center, H354 and H358 for Xenopus (6-4) PHR, whose mutations significantly lowered the enzymatic activity. Based upon highly sensitive FTIR analysis of the repair function, here we report that both H354A and H358A mutants of Xenopus (6-4) PHR still maintain their repair activity, although the efficiency is much lower than that of the wild type. Similar difference FTIR spectra between the wild type and mutant proteins suggest a common mechanism of repair in which (6-4) PP binds to the active center of each mutant, and is released after repair, as occurs in the wild type. Similar FTIR spectra also suggest that a decrease in volume by the H-to-A mutation is possibly compensated by the addition of water molecule( s). Such a modified environment is sufficient for the repair function that is probably controlled by proton-coupled electron transfer between the enzyme and substrate. On the other hand, two histidines must work in a concerted manner in the active center of the wild-type enzyme, which significantly raises the repair efficiency.
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