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

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.

Tryptophan-accelerated electron flow across a protein-protein interface.

We report a new metallolabeled blue copper protein, Re126W122Cu(I) Pseudomonas aeruginosa azurin, which has three redox sites at well-defined distances in the protein fold: Re(I)(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 Cu(I) oxidation (<50 ns), followed by slow back ET to regenerate Cu(I) and ground-state Re(I) 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, (Re126W122Cu(I))2 and that the forward ET is accelerated by intermolecular electron hopping through the interfacial tryptophan: *Re//←W122←Cu(I), 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 Cu(II) form shows two Re126W122Cu(II) 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 (Re126W122Cu(I))2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.

Expanding the molecular recognition repertoire of antifreeze polypeptides: effects on nucleoside crystal growth.

Despite differences in the crystal structures of ice and nucleosides, antifreeze polypeptides (AFPs) have been demonstrated to inhibit nucleation of 5-methyluridine, cytidine, and inosine and modify the crystal growth of the nucleosides efficiently. The molecular recognition repertoire of AFPs has been expanded to non-ice-like crystalline solids.