Electron tunneling in single crystals of Pseudomonas aeruginosa azurins

Rates of reduction of Os(III), Ru(III), and Re(I)* by Cu(I) in His83-modifi ed Pseudomonas aeruginosa azurins (M-Cu distance similar to 17 Angstrom) ha ve been measured in single crystals, where protein conformation and surface solvation are precisely defined by high-resolution X-ray structure determi nations: 1.7(8) x 10(6) s(-1) (298 K), 1.8(8) x 10(6) s(-1) (140 K), [Ru(bp y)(2)(im)(3+)-]; 3.0(15) x 10(6) s(-1) (298 K), [Ru(tpy)(bpy)(3+)-]; 3.0(15 ) x 10(6) s(-1) (298 K), [Ru(tpy)(phen)(3+)-]; 9.0(50) x 10(2) s(-1) (298 K ), [Os(bpy)(2)(iM)(3+)-]; 4.4(20) x 10(6) s(-1) (298 K), [Re(CO)(3)(phen)()*] (bpy = 2.2'-bipyridine, im = imidazole; tpy = 2,2':6'.2 " -terpyridine; phen = 1,10-phenanthroline). The time constants for electron tunneling in crystals are roughly the same as those measured in solution, indicating ver y similar protein structures in the two states. High-resolution structures of the oxidized (1.5 Angstrom) and reduced (1.4 Angstrom) states of Ru(II)( tpy)(phen)(His83)Az establish that very small changes in copper coordinatio n accompany reduction but reveal a shorter axial interaction between copper and the Gly45 peptide carbonyl oxygen [2.6 Angstrom for Cu(II)] than had b een recognized previously. Although Ru(bpy)(2)(im)(His83)Az is less solvate d in the crystal, the reorganization energy for Cu(I) --> Ru(III) electron transfer falls in the range (0.6-0.8 eV) determined experimentally for the reaction in solution. Our work suggests that outer-sphere protein reorganiz ation is the dominant activation component required for electron tunneling.