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

• Published in: ACS central science Vol. 5; no. 1; pp. 192 - 200
• Main Authors: Takematsu, Kana, Williamson, Heather R, Nikolovski, Pavle, Kaiser, Jens T, Sheng, Yuling, Pospíšil, Petr, Towrie, Michael, Heyda, Jan, Hollas, Daniel, Záliš, Stanislav, Gray, Harry B, Vlček, Antonín, Winkler, Jay R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.01.2019
• ISSN: 2374-7943; 2374-7951
• DOI: 10.1021/acscentsci.8b00882

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.