Hole Hopping Across a Protein-Protein Interface

We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCu^I, where two adjacent tryptophan residues (W124 and W122) are inserted between the Cu^I center and a Re photosensitizer coordinated to a H126 imidazole (Re = Re^I(H126)(CO)₃(dmp)⁺, dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (≤40 μM) triggers 70 ns electron transport over 23 Å, yielding a long-lived (120 μs) Re^I(H126)(CO)₃(dmp^•-)WWCu^II product. The Re126FWCu^I mutant (F124, W122) is not redox-active under these conditions. Upon increasing the concentration to 0.2-2 mM, {Re126WWCu^I}₂ and {Re126FWCu^I}₂ are formed with the dmp ligand of the Re photooxidant of one molecule in close contact (3.8 Å) with the W122' indole on the neighboring chain. In addition, {Re126WWCu^I}₂ contains an interfacial tryptophan quadruplex of four indoles (3.3-3.7 Å apart). In both mutants, dimerization opens an intermolecular W122' → //*Re ET channel (// denotes the protein interface, *Re is the optically excited sensitizer). Excited-state relaxation and ET occur together in two steps (time constants of ∼600 ps and ∼8 ns) that lead to a charge-separated state containing a Re(H126)(CO)₃(dmp^•-)//(W122^•+)' unit; then (Cu^I)' is oxidized intramolecularly (60-90 ns) by (W122^•+)', forming Re^I(H126)(CO)₃(dmp^•-)WWCu^I//(Cu^II)'. The photocycle is closed by ∼1.6 μs Re^I(H126)(CO)₃(dmp^•-) → //(Cu^II)' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 μs step in Re126WWCu^I. Importantly, dimerization makes Re126FWCu^I photoreactive and, as in the case of {Re126WWCu^I}₂, channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of Re^I(H126)(CO)₃(dmp^•-)//Cu^II. Although two adjacent W124 and W122 indoles dramatically enhance Cu^I → *Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCu^I}₂ does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCu^II}₂ or {Re126FWCu^II}₂ also triggers intermolecular W122' → //*Re ET, and the Re(H126)(CO)₃(dmp^•-)//(W122^•+)' charge-separated state decays to the ground state by ∼50 ns Re^I(H126)(CO)₃(dmp^•-)⁺ → //(W122^•+)' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport.