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*) induces an electron transfer over approximately 15 A from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH* <-- W382 <-- W359 <-- W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH* decayed with a time constant tau approximately 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (tau approximately 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH* is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield ( approximately 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* is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH* (tau approximately 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* in WT and from W359 to excited FADH* in W382F mutant photolyase were estimated and related to the donor-acceptor distances.