Electron Transfer in the Cytochrome c - Peroxidases Complex

细胞色素c-细胞色素c过氧化物酶（CcP）系统提供了一个非常丰富的机会来研究蛋白质-蛋白质相互作用和生理相关血红素蛋白之间的远程电子转移的亲密细节。本研究计划的目标在于两个方面。第一个领域是表征马和酵母的iso1细胞色素c与酵母CcP之间的相互作用。实验旨在阐明细胞色素c和CcP之间结合的化学计量学，以确定是否以及在什么条件下，多个细胞色素c可以与CcP结合。高亲和力细胞色素c在CcP上的结合位点将通过定点诱变来定位。单个氨基酸在结合位点的作用将通过结合亲和力的热力学测量来评估。任何二级相互作用位点的位置也将通过位点定向诱变确定。第二个感兴趣的领域是表征细胞色素c和CcP血红素基团之间的电子转移。瞬态动力学技术将用于研究从铁细胞色素c到CcP氧化中间体的电子转移速率以及CcP化合物II中血红素铁和Trp- 191之间的氧化还原平衡速率。通过对CcP的定点诱变，探索反应动力学的特异性和电子传递途径。这项工作将有助于更好地理解蛋白质识别，特别是血红素蛋白，包括血红蛋白和能量代谢中的蛋白质

The cytochrome c-cytochrome c peroxidase (CcP) system provides an exceedingly rich opportunity to investigate the intimate details of protein-protein interactions and long-range electron transfer between physiologically related heme proteins. The objectives of this research proposal lie in two areas. This first area is to characterize the interaction between both horse and yeast iso-1 cytochrome c with yeast CcP. Experiments designed to clarify the stoichiometry of binding between cytochrome c and CcP will be performed to determine if, and under what conditions, more than one cytochrome c may bind to CcP. The high-affinity cytochrome c binding site on CcP will be mapped through site-directed mutagenesis. The role of individual amino acids at the binding site will be assessed through thermodynamic measurements of the binding affinity. The location of any secondary interaction sites will also be determined through site-directed mutagenesis. The second area of interest is to characterize electron transfer between the heme groups of cytochrome c and CcP. Transient-state kinetic techniques will be used to investigate the rate of electron transfer from ferrocytochrome c to the oxidized intermediate of CcP and the rate of redox equilibration between the heme iron and Trp- 191 in CcP compound II. The specificity of the reaction dynamics and the electron transfer pathway will be explored through site- directed mutagenesis of CcP. %%% This work should lead to a better understanding of protein-protein recognition in general, and of heme proteins, which include hemoglobin and the proteins in energy metabolism, in particular