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

[Image: see text] 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.