Ultrafast Cycloreversion\nof Thymine-Toluene [2 + 2]\nCycloadducts by DNA Photolyase

DNA photolyases use blue light and fully reduced flavin\ncofactor\nto repair UV-induced cyclobutane pyrimidine dimers (CPD) formed between\ntwo adjacent thymine bases in DNA. Thymine can form [2 + 2] cyclobutane\nadducts with its biological isosteres like toluene upon UV irradiation,\nresulting in chemically different analogues of CPD. Here, we investigated\nthe cycloreversion reactions of two such adducts formed between thymine\nand toluene, T<>Tol, catalyzed by a class-I CPD photolyase.\nThe\nphotolyase can bind to the T<>Tol adducts efficiently and restore\nthe constituent bases upon excitation. Using femtosecond spectroscopy,\nwe systematically characterized all the elementary steps involved\nin the enzymatic cycloreversion of the T<>Tol adducts and comprehensively\nanalyzed the key intermolecular electron-transfer (ET) reactions and\ncyclobutane bond splitting steps. The initial electron injection to\nthe bound adducts happens primarily through a two-step electron hopping\nmechanism, unlike in CPD repair where direct electron tunneling is\ndominant. After electron injection and ultrafast first-bond splitting,\nthe delicate competition between the second bond splitting and a futile\nback ET dictates the overall reaction quantum yields of the adducts,\ninfluenced by the stability of adduct intermediates and steric crowding\naround the constituent bases. The final electron return for the cycloreversion\nreactions adopts a different pathway compared to CPD repair. The photolyase\nutilizes its conserved photorepair mechanism and allows ET pathway\nflexibility to reverse the [2 + 2] cycloaddition reaction of non-natural\nanalogues of CPD.