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

Abstract

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.