Kinetics of inter-protein electron transfer between the model system cytochrome c peroxidase and cytochrome c

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc) form a complex system because CcP contains two redox centers: the heme and an adjacent tryptophan (W191). These protein partners occupy a central role in the study of interprotein ET. The heme of either partner can be modified to exhibit photo-initiated ET through substitution of Fe with Zn (or Mg). Laser-excitation of the Zn-porphyrin (ZnP) to its triplet excited state initiates direct heme-heme ET to the fern-heme center across the protein-protein interface; this produces a charge-separated intermediate which returns to the ground state by a thermal ET process. Variants of wild type (WT) CcP were used to study the effects of key protein residues on binding and reactivity with Cc.; A mutation of tryptophan 191 to an unreactive phenylalanine shows an increased signal from the ET intermediate without affecting forward ET kinetics. The studies demonstrate that W191 provides an alternate ('short-circuit') pathway for back ET with Fe2+Cc that is much faster than back ET with Zn+·CcP. We extended this study to a variant of CcP where five of the tryptophan and tyrosine residues near the heme were mutated to phenylalanine (WYM4). The redox chemistry of ZnCcP and its redox mutants W191F and WYM4 is studied to further understand the role of oxidizable amino acids in ET and in the Cc binding process.; The effects of temperature, viscosity, pH, and ionic strength on ET were also studied. When examined as a function of temperature and viscosity, the forward ET rate decreases while the back ET rates initially increase with viscosity at fixed temperature. This shows that dynamic motions facilitate forward ET but can suppress back ET.; The protein system was also studied as a trapped, bound complex within the matrix of optically transparent silicate gels. The gel had no affect on the quenching kinetics of the system or the structure of the proteins. Triplet anisotropy measurements show the majority of the proteins to be freely rotating in the sol-gels as reactive complexes. Conditions of ionic strength, pH, and/or glycerol were controlled and altered to change the aqueous environment of the protein complex.