Electron Transfer in the Cytochrome c - Cytochrome c Peroxidase Complex

细胞色素c -细胞色素c过氧化物酶系统为揭示过氧化物酶活性的结构要求以及生理相关血红素蛋白之间的长距离电子转移提供了一个绝佳的机会。本研究计划的目的是阐明细胞色素c过氧化物酶催化的详细机制，特别强调细胞色素c与细胞色素c过氧化物酶的相互作用。实验旨在阐明细胞色素c和细胞色素c过氧化物酶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可以与细胞色素c过氧化物酶结合。我们将利用从不同来源分离的多种细胞色素cs，并通过细胞色素c过氧化物酶的定点诱变来探索高亲和力结合位点的特异性。瞬态动力学技术将用于研究铁细胞色素c和细胞色素c预氧化酶的氧化中间体之间的电子转移速率。反应动力学的特异性将通过使用来自不同来源的细胞色素c来评估，特异氨基酸残基在酶中的作用将通过位点定向诱变来探索。

The cytochrome c - cytochrome c peroxidase system provides an outstanding opportunity to uncover the intimate details of the structural requirements for peroxidase activity, as well as those for long range electron transfer between physiologically related heme proteins. The objective of this research proposal is to elucidate the detailed mechanism of cytochrome c peroxidase catalysis with special emphasis on characterizing the interaction between cytochrome c and cytochrome c peroxidase. Experiments designed to clarify the stoichiometry of binding between cytochrome c and cytochrome c peroxidase will be performed to determine if, and under what conditions, more than one cytochrome c may bind to cytochrome c peroxidase. The specificity of binding at the high affinity binding site will be explored by utilizing a variety of cytochrome cs isolated from different sources and through site-directed mutagenesis of cytochrome c peroxidase. Transient state kinetic techniques will be used to investigate the rate of electron transfer between ferrocytochrome c and the oxidized intermediates of cytochrome c preoxidase. The specificity of the reaction dynamics will be evaluated by using cytochrome c from various sources and the role of specific amino acid residues in the enzyme will be explored by site-directed mutagenesis.