Two Tryptophans Are Better Than One in Accelerating Electron Flow through a Protein

We have constructed and structurally characterized a Pseudomonas aeruginosa azurin mutant Re126WWCu I , where two adjacent tryptophan residues (W124 and W122, indole separation 3.6-4.1 Å) are inserted between the Cu I center and a Re photosensitizer coordinated to the imidazole of H126 (Re I (H126)(CO) 3 (4,7-dimethyl-1,10-phenanthroline) + ). Cu I oxidation by the photoexcited Re label (∗Re) 22.9 Å away proceeds with a ∼70 ns time constant, similar to that of a single-tryptophan mutant (∼40 ns) with a 19.4 Å Re-Cu distance. Time-resolved spectroscopy (luminescence, visible and IR absorption) revealed two rapid reversible electron transfer steps, W124 →∗Re (400-475 ps, K 1 3.5-4) and W122 → W124 •+ (7-9 ns, K 2 0.55-0.75), followed by a rate-determining (70-90 ns) Cu I oxidation by W122 •+ ca. 11 Å away. The photocycle is completed by 120 μs recombination. No photochemical Cu I oxidation was observed in Re126FWCu I , whereas in Re126WFCu I , the photocycle is restricted to the ReH126W124 unit and Cu I remains isolated. QM/MM/MD simulations of Re126WWCu I indicate that indole solvation changes through the hopping process and W124 →∗Re electron transfer is accompanied by water fluctuations that tighten W124 solvation. Our finding that multistep tunneling (hopping) confers a ∼9000-fold advantage over single-step tunneling in the double-tryptophan protein supports the proposal that hole-hopping through tryptophan/tyrosine chains protects enzymes from oxidative damage