Syntheses of novel ruthenium-cytochrome c complexes and double mutant studies of cytochrome c and cytochrome c oxidase interaction with flash photolysis technique

Millett and Durham research groups have developed a laser flash photolysis method that uses photoactive ruthenium complexes to measure fast electron transfers (ET) in the bacterial and mitochondrial respiratory chains. Explored here are the syntheses of two covalently attached ruthenium complexes whose efficiency of cytochrome c (Cc) photoreduction and photooxidation is larger than that of the conventional tris(bipyridine)ruthenium(II) complex. By means of Ru(I) formation, [Ru(bpz)2(dmb)]-C c derivative {lcub}bpz=2,2'-bipyrazine-N,N'; dmb=4,4'-dimethyl-2,2'-bipyridine-N,N'{rcub} is advantageous for intermolecular photooxidation studies with 20% yield of photooxidized Cc generated in a single flash, having a rate constant of (1.5+/-0.2)x106 s-1 , whereas none was ever observed with the conventional ruthenium. Leaning towards usefulness in intermolecular photoreduction studies, [Ru(bpd) 2(dmb)]-Cc {lcub}bpd=3,3'-bipyridazine-N,N'{rcub} generates heme c photoreduction at a rate of (1.0+/-0.2)x105 s-1 with 22% yield, a six-fold increase from the conventional ruthenium.; [Ru(bpz)2(dmb)]2+ reagent allows for the first time measurement of the true intracomplex ET in the forward, physiological direction (heme c1 to Cc with a rate constant of 1.4x104 s-1) in the yeast cytochrome bc1:Cc complex, where hydrophobic interactions may play an important role. Intermolecular photooxidation studies of rat cytochrome b5 and yeast [Ru(bpz)2(dmb)]-Cc capture ET rate of 10 5 s-1 from heme b5 to heme c, while ET rate of (1.0+/-0.2)x104 s -1 is observed from cytochrome c1 to [Ru(bpz) 2(dmb)]-labeled cytochrome c552 in P. denitrificans system. Characterization of ET kinetics in beef cytochrome c oxidase (CcO) using yeast [Ru(bpd) 2(dmb)]-Cc demonstrates ET rates of 60,000 s -1 and 20,000 s-1 from heme c to CU A and subsequently to heme a, respectively.; Also investigated is the characterization of horse Cc and R. sphaeroides CcO interactions by double mutant analysis, which supports a two-step model for complex formation. The rate of ET between the two native proteins is (5.5+/-0.7)x10 4 s-1. The effects of the mutants indicated that C cO negatively charged residues (D214, E157, D195, and E148) and C c positively charged residues (K13, K87, K86, and K27) contributed to the interaction between the two proteins. The cooperative interactions of these residues contribute to the proteins' surface potential, which drives the docking of Cc onto CcO. Perhaps the actual CcO:Cc complex promotes nonexclusive and unspecific pairwise electrostatic interactions.