EPR AND RESONANCE RAMAN SPECTROSCOPY

本核心中描述的光谱学研究的长期目标是 项目是利用从X射线获得的结构信息 前列腺素H合酶和细胞色素的晶体学研究 c氧化酶（CcOX）作为探索其催化细节的平台 机制，并开发新的光谱方法进行研究， 生物系统中的金属中心。振动光谱学，共振 拉曼和傅立叶变换红外光谱，提供有关键的详细信息 强度，将扩展到能量制度，其中金属配体键合 当与小容量、停流 动力学技术，这些光学方法提供了一个独特的途径， 表征催化中间体。先进的EPR方法， 在这个核心项目中使用，允许一个测量波函数 它包含了一个不成对的电子自旋， 催化位点的电子结构。EPR开发工作， 将在这个核心项目中进行，包括开发一个 提高我们能力的脉冲微波/脉冲磁场方法 为了解决高自旋顺磁金属中心的超精细耦合， 几种新的相干转移脉冲EPR方法在 蛋白质自由基构象测定与配体超精细 耦合，并并行开发分析策略，以支持 这些测量。因为在这篇文章中描述的开发工作 提案由CcOX解决的具体问题驱动， PGHS，在本项目过程中定义的方法将是 对生物医学界普遍有用。

The long-range goal of the spectroscopic studies described in this core project are to use the structural information gained from X-ray crystallographic studies of Prostaglandin H Synthase (PGHS) and Cytochrome c Oxidase (CcOX) as platforms for exploring the details of their catalytic mechanisms, and to develop new spectroscopic methods for the study of metal centers in biological systems. Vibrational spectroscopies, Resonance Raman and FTIR, which provide detailed information regarding bond strengths, will be extended to energy regimes where metal-ligand bonding can be better characterized When combined with small-volume, stop-flow kinetic techniques, these optical methods provide a unique pathway for characterizing catalytic intermediates. The advanced EPR methods that will be utilized in this core project, allow one to measure to wave function that house an unpaired electron spin and gain an understanding of the electronic structure of a catalytic site. The EPR development work that will be undertaken in this core project includes the development of a pulse-microwave/pulse-magnetic field method for improving our capability to resolve hyperfine couplings for high-spin paramagnetic metal centers, the application of several new coherence transfer pulsed EPR methods for the measurement of protein radical conformation and ligand hyperfine couplings, and the parallel development of analysis strategies to support these measurements. Because the development work described in this proposal is driven by specific questions being addressed for CcOX and PGHS, the methodologies defined during the course of this project will be generally useful to the biomedical community.