Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: Toward a detailed understanding of the triple tryptophan electron transfer chain

The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382-W359-W306 in photolyase from E. coli) that bridges the ∼15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH -; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH-. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond-nanosecond time window bridging the time domains covered by ultrafast pump-probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH- W306°+ was found at 19 ± 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+. With this yield, the optical spectrum of the excited state of FADH° can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain.

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