Role of the Middle Residue in the Triple Tryptophan Electron Transfer Chain of DNA Photolyase:  Ultrafast Spectroscopy of a Trp→Phe Mutant

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH• in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH•−W382−W359−W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH•* was found to decay in ∼30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH• fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382•+ and (in WT) electron transfer from W359 to W382•+ occur with time constants