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, K₁ ≅ 3.5–4) and W122 → W124^•+ (7–9 ns, K₂ ≅ 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.

Photoactive charge transfer compounds are of strong interest for their potential applications in material, chemical, and biological science and their abilities to elucidate fundamental charge transfer mechanisms. Aminonaphthols, photoacids with both oxygen (OH) and nitrogen-based (NH₂) protonation sites, have been reported to undergo simultaneous excited-state proton transfer (ESPT) in water upon excitation. In this paper, the ESPT mechanism for zwitterion formation in 8-amino-2-naphthol (8N2OH) and 5-amino-2-naphthol (5N2OH) was examined using a combination of time-resolved emission spectroscopy and time-dependent density functional theory (TD-DFT) calculations. The measurements prompted a re-assignment of the zwitterion state in the steady-state emission spectra; analysis of the time-correlated single-photon counting emission data showed that the zwitterion was formed only from excitation of protonated 5N2OH and 8N2OH such that ESPT occurred only at the single hydroxyl group. The protonation state of the amino group dramatically altered the photoacidity of OH, such that the pH behaved as an on/off switch for photoacidity. In the protonated state (NH₃ ⁺), the pK_a*(OH) values of 5N2OH and 8N2OH were both 1.1 ± 0.2, while in the deprotonated state (NH₂), the two pK_a*(OH) values were similar to the ground state proton acidity, pK_a(OH) = 9.5 ± 0.2. The switching of the photoacidity was investigated using TD-DFT calculations and the linear free energy Hammett relation. The latter was shown to not describe the excited state data over the broad pH range.