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

We report a new metallolabeled blue copper protein, Re126W122CuI Pseudomonas aeruginosa azurin, which has three redo sites at well-defined distances in the protein fold: ReI(CO)3(4,7-dimethyl-1,10-phenanthroline) covalently bound at H126, a Cu center, and an indole side chain W122 situated between the Re and Cu sites (Re-W122(indole) = 13.1 Å; dmp-W122(indole) = 10.0 Å, Re-Cu = 25.6 Å). Near-UV excitation of the Re chromophore leads to prompt CuI oxidation (<50 ns), followed by slow back ET to regenerate CuI and ground-state ReI with biexponential kinetics, 220 ns and 6 μs. From spectroscopic measurements of kinetics and relative ET yields at different concentrations, it is likely that the photoinduced ET reactions occur in protein dimers, (Re126W122CuI)2, and that the forward ET is accelerated by intermolecular electron hopping through the interfacial tryptophan: *Re//←W122←CuI, where // denotes a protein-protein interface. Solution mass spectrometry confirms a broad oligomer distribution with prevalent monomers and dimers, and the crystal structure of the CuII form shows two Re126W122CuII molecules oriented such that redox cofactors Re(dmp) and W122-indole on different protein molecules are located at the interface at much shorter intermolecular distances (Re-W122(indole) = 6.9 Å, dmp-W122(indole) = 3.5 Å, and Re-Cu = 14.0 Å) than within single protein folds. Whereas forward ET is accelerated by hopping through W122, BET is retarded by a space jump at the interface that lacks specific interactions or water molecules. These findings on interfacial electron hopping in (Re126W122CuI)2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.

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 pKa*(OH) values of 5N2OH and 8N2OH were both 1.1 ± 0.2, while in the deprotonated state (NH2), the two pKa*(OH) values were similar to the ground state proton acidity, pKa(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.