Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

Abstract

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH^•) induces an electron transfer over ≈15 Å 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 τ ≈ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (τ ≈ 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 (≈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^• (τ ≈ 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.