Tryptophan-Accelerated Electron Flow Across a Protein-Protein Interface

摘要

We report a new metallolabeled blue copper protein, Re126W122Cu~I Pseudomonas aeruginosa azurin, which has three redox sites at well-denned 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 A, dmp-W122(indole) = 10.0 A, Re-Cu = 25.6 A). 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 comers,(Rel26W122Cu~I)_2 and that the forward ET is accelerated by intermolecular electron hopping through the interracial 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 Rel26W122Cu~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 A, dmp-W122(indole) = 3.S A, and Re-Cu = 14.0 A) 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 (Rel26W122Cu~Ⅰ)_2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes.