Dissection of the Triple Tryptophan Electron Transfer Chain in Escherichia coli DNA Photolyase: Trp382 Is the Primary Donor in Photoactivation

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state$(FADH^\bullet)$induces an electron transfer over$\approx\!15\>\AA$from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain$FADH^\bullet \leftarrow W382 \leftarrow W359 \leftarrow W306$. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited$FADH^\bullet$decayed with a time constant$\tau \approx 26\>ps$to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived$(\tau \approx 80\>ps)$, and no significant amount of product was detected. We conclude that, in WT photolyase, excited$FADH^\bullet$is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (≈0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited$FADH^\bullet$is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited$FADH^\bullet\>(\tau \approx 80\>ps)$, explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited$FADH^\bullet$in WT and from W359 to excited$FADH^\bullet$in W382F mutant photolyase were estimated and related to the donor-acceptor distances.