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是一种分子量为34,000的单体血红素蛋白，以细胞色素c为电子供体，催化过氧化氢还原裂解为水。解决了CCP、其两电子氧化产物CCP、化合物I(酶与过氧化氢反应形成的化合物I)、酶：抑制剂络合物以及几个突变的CCP的晶体结构。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。拟议的研究将使用定点突变来扰乱酶的结构，并使用X射线结晶学来描述由此产生的结构变化。随后将使用光谱和动力学技术对这些突变体进行表征，以将结构的变化与酶的初级反应的变化联系起来。拟议的研究结果将为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供洞察。这项拟议的工作还将深入了解蛋白质用来修饰血红素假体基团的反应性以完成特定反应的机制。

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.