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上相关的血红素蛋白之间的远程电子转移的细节。这项研究提案的目标在于两个方面。这第一个领域是描述马和酵母iso-1细胞色素c与酵母CCP之间的相互作用。将进行旨在阐明细胞色素c与CCP之间结合的化学计量学的实验，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。CCP上的高亲和力细胞色素c结合位点将通过定点突变进行定位。单个氨基酸在结合部位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定点突变来确定。第二个感兴趣的领域是表征细胞色素c和CCP的血红素基团之间的电子转移。我们将利用暂态动力学技术研究CCP化合物II中铁细胞色素c向CCP氧化中间体的电子转移速率，以及CCP化合物II中铁血红素铁与Trp-191之间的氧化还原平衡速率。这项工作应该有助于更好地理解蛋白质-蛋白质识别，特别是血红素蛋白质，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