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 mu M) triggers 70 ns electron transport over 23 angstrom, yielding a long-lived (120 mu s) Re-I(H126)(CO)(3)(dmp(center dot-))WWCuII 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 angstrom) 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 angstrom 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 similar to 600 ps and similar to 8 ns) that lead to a charge-separated state containing a Re(H126)(CO)(3)(dmp(center dot-))//(W122(center dot+))' unit; then (Cu-I)' is oxidized intramolecularly (60-90 ns) by (W122(center dot+))', forming Re-I(H126)(CO)(3)(dmp(center dot-))WWCuI//(Cu-II)'. The photocycle is closed by similar to 1.6 mu s Re-I(H126)(CO)(3)(dmp(center dot-)) -> //(Cu-II)' back ET that occurs over 12 angstrom, in contrast to the 23 angstrom, 120 mu s step in Re126WWCu(I). Importantly, dimerization makes Re126FINCu(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)(COI)(3)(dmp(center dot-))//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 {Re1264VINCu(II)}(2) or {Re126FWCu(II)}(2) also triggers intermolecular W122' -> //*Re ET, and the Re(H126)(CO)(3)(dmp(center dot-))// (W122(center dot+))' charge-separated state decays to the ground state by similar to 50 ns Re-I(H126)(CO)(3)(dmp(center dot-))(+) -> //(W122(center dot+))' 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.