​Abstract

Class II DNA photolyases are flavoenzymes occurring in both prokaryotes and eukaryotes including higher plants and animals. Despite considerable structural deviations from the well-studied class I DNA photolyases, they share the main biological function, namely light-driven repair of the most common UV-induced lesions in DNA, the cyclobutane pyrimidine dimers (CPDs). For DNA repair activity, photolyases require the fully reduced flavin adenine dinucleotide cofactor, FADH⁻, which can be obtained from oxidized or semi-reduced FAD by a process called photoactivation. Using transient absorption spectroscopy, we have examined the initial electron and proton transfer reactions leading to photoactivation of the class II DNA photolyase from Methanosarcina mazei. Upon photoexcitation, FAD is reduced via a distinct (class II-specific) chain of three tryptophans, giving rise to an FAD˙⁻ TrpH˙⁺ radical pair. The distal Trp₃₈₈H˙⁺ deprotonates to Trp₃₈₈˙ in 350 ps, i.e., by three orders of magnitude faster than TrpH˙⁺ in aqueous solution or in any previously studied photolyase. We identified a class II-specific cluster of protein-bound water molecules ideally positioned to serve as the primary proton acceptor. The high rate of Trp₃₈₈H˙⁺ deprotonation counters futile radical pair recombination and ensures efficient photoactivation.

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