We have constructed and structurally characterized a Pseudomonas aeruginosa azurin mutant Re126WWCuI , where two adjacent tryptophan residues (W124 and W122, indole separation 3.6-4.1 Å) are inserted between the CuI center and a Re photosensitizer coordinated to the imidazole of H126 (ReI(H126)(CO)3(4,7-dimethyl-1,10-phenanthroline)+). CuI 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) CuI oxidation by W122•+ ca. 11 Å away. The photocycle is completed by 120 μs recombination. No photochemical CuI oxidation was observed in Re126FWCuI , whereas in Re126WFCuI , the photocycle is restricted to the ReH126W124 unit and CuI remains isolated. QM/MM/MD simulations of Re126WWCuI 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.

Beckman Institute California Institute of Technology Pasadena California 91125 United States

Central Laser Facility Research Complex at Harwell Science and Technology Facilities Council Rutherford Appleton Laboratory Harwell Oxford Didcot Oxfordshire OX11 0FA U K

Department of Chemistry Bowdoin College Brunswick Maine 04011 United States

Department of Chemistry Xavier University of Louisiana New Orleans Louisiana 70125 United States

Department of Physical Chemistry University of Chemistry and Technology Prague Technická 5 CZ 166 28 Prague Czech Republic

J Heyrovský Institute of Physical Chemistry Academy of Sciences of the Czech Republic Dolejškova 3 CZ 182 23 Prague Czech Republic

School of Biological and Chemical Sciences Queen Mary University of London Mile End Road London E1 4NS United Kingdom

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Tryptophan to Tryptophan Hole Hopping in an Azurin Construct

Photoinduced hole hopping through tryptophans in proteins

Hole Hopping Across a Protein-Protein Interface