Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain
DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH°. The catalytically potent state FADH-can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH° is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 Å distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH°, in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH°* ← W382 ← W359 ← W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH° and leads to the formation of FADH-and a deprotonated tryptophanyl radical, most likely W359°. These photoproducts are formed in less than 10 ns and recombine to the dark state in ∼1 μs. These results support the electron hopping mechanism.