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

Funding Source

Arnold and Mabel Beckman Foundation, National Institutes of Health, Gordon and Betty Moore Foundation, Grantová agentura České republiky, National Institute of Diabetes and Digestive and Kidney Diseases, Science and Technology Facilities Council

Grant Number

R01DK019038, 17-011375


Department of Chemistry

Document Type


Publication Date



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.


DOI: 10.1021/acscentsci.8b00882

Funding text

We thank Martin Pizľ (JH Institute) for his help analyzing the TRIR spectra. Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number R01DK019038. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support was provided by the Arnold and Mabel Beckman Foundation, the Czech Science Foundation (GAČ R) Grant 17-011375, and the STFC Rutherford Appleton Laboratory (UK). X-ray crystallography data were collected on SSRL Beamline 12-2 through the support of the Caltech Molecular Observatory, funded by the Gordon and Betty Moore Foundation, Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program. Operations at SSRL are supported by U.S. DOE and NIH.