Crystallographic And Spectroscopic Studies On The Complex Of Cytochrome C Peroxidase And Cytochrome C

Abstract

Electron transfer (ET) is a ubiquitous process that underlies the physics of biology. Photosynthesis, respiration, and metabolism all require careful movement of electrons across long distances to generate the energy and chemical compounds necessary for life. Most electron transfer is controlled by proteins, which guide electrons to their destination through small jumps along a carefully arranged network of cofactors. Cytochrome c Peroxidase(CcP) is a yeast protein that converts hydrogen peroxide to water, in a reaction catalyzed by reduction from Cytcochrome c (Cc). In the process of converting hydrogen peroxide to water, CcP generates a tryptophanyl radical on W191. This radical acts a “hopping site” for electron transfer, bridging the gap as a stepping stone between the hemes of Cc and CcP. In this study, we show that tyrosine (W191Y) can act as an effective hopping site in the same location, but that phenylalanine (W191F) cannot. Through transient absorption spectroscopy, we measure the rates of photoinitiated electron transfer from Zn-substituted CcP to Cc and back, and determine the rates of ET from Cc to W191F CcP, where the ET rates are such that the charged-separated intermediate can be observed. From these measurements, we calculate the reorganization energy of the reaction and determine that ET falls in the Marcus inverted region. We also observe the effect of ionic strength on complex formation and ET kinetics. Through X-ray crystallography, we solved the structure of the various CcP mutants in complex with Cc, including the W191G mutation, which creates an empty cavity where other small compounds will bind. We showed that these compounds, although similar in structure to tryptophan and tyrosine, do not rescue peroxidase activity, and conclude that a hopping site must not only have the correct potential, but also be held stably in position and perhaps even covalently bonded to the electron acceptor.