| The characterization of electron transfer reactions involving cytochrome bc1 was studied using the ruthenium laser flash photolysis technique. In conjunction with this study, [Ru(bpz)2]2(qpy)(PF 6)4 {bpz, 3,3'-bipyrazine; qpy, 2,2':4',4";2",2"'-quaterpyridine} was developed. This binuclear ruthenium complex has a charge of +4, with a lifetime of the excited state at 0.4 μs. From preliminary studies with several metalloproteins, this complex was shown to increase photoreduction yields significantly over other relevant ruthenium complexes.;Early observation showed that yeast Ruz-39-Cc {Cc, cytochrome c) has stronger nonpolar interactions than horse Ruz39-Cc. Several mutants involving the residues along the interacting surface of yeast Cc were generated to characterize the residues involved in the interactions with cytochrome c1. Experimentally, the single sulfhydryl on the backside of yeast H39C;C102T iso-1-Cc was labeled with [Ru(2,2'-bipyrazine) 2(4-bromomethyl-4'-methyl-2,2'-bipyridine)]2+ to form Ruz-39-Cc. A single laser flash of a reduced yeast cytochrome bc1 : Ruz-39-C c complex results in the intercomplex electron transfer rate from c1 to c, which was observed at various ionic strengths. Applying the same technique, the kinetic of the yeast C c mutants were measured at different ionic strengths. The results confirmed the significance of the strong planar interaction between Arg-13 of yeast Cc and Phe-230 of yeast cytochrome c 1.;X-ray crystallographic studies of cytochrome bc 1 have revealed that famoxadone binding leads to significant conformation changes along the EF loop residues in the cytochrome b subunit. Consequently, an extensive number of R. sphaeroides cytochrome bc1 mutants were designed involving these residues to probe the relevance of EF loop structural changes to iron-sulfur protein (ISP) movement. The kinetics of wild-type cytochrome bc1 were measured using [Ru(bpy)2]2(qpy)(PF6) 4 {bpy, 2,2'-bipyridine} to rapidly photooxidize cytochrome c1, followed by electron transfer from ISP to cytochrome c1 with a rate constant k1 of 60,000 s-1, which is gated by ISP movement. Oxidant-induced reduction of cytochrome bH occurred with a rate constant k2 of 2,300 s-1, which is rate-limited by electron transfer from quinol to ISP. These kinetic parameters were obtained from the mutants, and then compared with those parameters obtained in the presence of famoxadone. This study suggests that EF loop plays a role in the regulation of ISP movement. |
