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

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... roduct. 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 chargeseparated 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 . Whereas 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 interand 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. Graphical Abstract Takematsu et al. Cu II forms of Re126FWCu, Re126WWCu, and Re126WFCu at concentrations 200 μM and higher, aiming to shed light on the interplay between intraand intermolecular electron/ hole hopping. RESULTS Structures. The X-ray crystal structure of Re126WWCu II (PDB: 6MJS; Figure 1A ) 21 shows multiple short (3.5 -4.0 Å) intramolecular contacts between the mutually T-oriented aromatic groups of redox cofactors Re(H126)(CO) 3 (dmp) + , W124, and W122 along the 22.9 Å intramolecular Re---Cu ET pathway (see Table S1 for ET-relevant intramolecular distances). 21 The W122 indole is separated from the Cu atom by ~11 Å; and the structure of the W122-Cu pathway is virtually the same as in Re126FWCu II , 21 Re126T124W122Cu II , 22 and Takematsu et al. applicable to Re126WFCu I (k 5 and all subsequent rate constants = 0) and Re126FWCu I (k 1 and all subsequent rate constants = 0; (k 4 ) −1 is the unquenched 3 CT lifetime, 1.15 μs.) Takematsu et al.