Crystallographic and Mechanistic Studies of Cytochrome c Peroxidase

本研究的长期目标是了解细胞色素c过氧化物酶（CCP）的结构和催化机制。CCP是一种34000毫瓦特的单体血红素蛋白，使用亚铁细胞色素c作为电子供体，催化过氧化氢还原裂解成水。CCP的晶体结构，它的双电子氧化产物，化合物I（由酶与过氧化物反应形成），酶：抑制剂配合物，和几个突变的CCP已经解决。可用的结构信息使CCP成为研究血红素蛋白分子间电子转移和O-O键激活的一个有吸引力的系统。拟议的研究将采用定点诱变来扰乱酶的结构和x射线晶体学来描述由此产生的结构变化。通过光谱和动力学技术对这些突变体的后续表征将用于将结构变化与酶的主要反应变化联系起来。提出的研究结果将提供深入了解大分子之间的静电识别和血红素蛋白之间的电子快速转移所涉及的结构特征。提出的工作也将提供深入了解的机制，蛋白质用来修改血红素假体基团的反应性，以完成一个特征性的反应。

The long term goal of this research is to understand the structure and catalytic mechanism of cytochrome c peroxidase (CCP). CCP is a 34,000 M.W. monomeric heme protein that catalyses the reductive cleavage of peroxide to water, using ferrous cytochrome c as the electron donor. The crystal structures of CCP, its two-electron oxidation product, Compound I (formed by the reaction of the enzyme with peroxide), enzyme:inhibitor complexes, and several mutant CCPs have been solved. The available structural information makes CCP an attractive system for the study of intermolecular electron transfer and O-O bond activation by heme proteins. The proposed investigation will employ site-directed mutagenesis to perturb the enzyme structure and X-ray crystallography to describe the resulting structural changes. Subsequent characterization of these mutants by spectroscopic and kinetic techniques will be used to relate the changes in structure to changes in the primary reactions of the enzyme. The results of the proposed research will provide insight into the structural features that are involved in electrostatic recognition between macromolecule and rapid transfer of electrons between heme proteins. The proposed work will also provide insight into the mechanisms that proteins use to modify the reactivity of the heme prosthetic group to accomplish a characteristic reaction.