DISTANCE MEASUREMENTS IN METALLOPROTEINS

在呼吸和光合作用中的能量转换涉及 将电子转移反应与生成 电化学梯度穿过双层膜。尽管序列 电子转移反应的动力学基本上是已知的 这些系统，更少的是知道的空间布局， 电子载体和它们之间的距离。理想情况下，电子 载体可以用作探测器， 组织和远程电子转移的距离， 生物电子传递过程。长期目标是 项目是使用电子自旋弛豫增强测量， 确定蛋白质中氧化还原活性位点之间的距离并进行探测 多核金属离子团簇的磁性。第一 目的是表征成对磁相互作用的影响 电子自旋弛豫率的样品中，距离 相互作用的中心和每个中心的磁性是 知道的在这项研究中，反应中心蛋白Rb。sphaeroides 将被用作模型系统。这种蛋白质可以在一种状态下制备 它包含两个固定距离为28埃的顺磁位点。 细菌叶绿素电子自旋弛豫速率的增强 由于与非血红素的磁相互作用而产生的特殊对阳离子自由基 Fe 2+，或Mn 2+或Cu 2+在Fe 2+位，将被系统地 研究通过改变金属离子和温度在两个非- 定向和定向样品。这些数据将用于确定 的适用性限制，并提供数据，以扩大， 自旋弛豫增强理论第二个目标是使用 电子自旋晶格弛豫速率增强的测量， 确定氧化还原位点的空间组织和磁性 在细胞色素c氧化酶、光系统II和一氧化氮合酶中， 结构信息不可用的系统。两种类型的 将继续进行应用，以获得结构和磁性 在这三个系统中的信息，并建立一般 这种方法的适用性，以确定距离和磁数据， 其他系统。第一个应用是测量自旋-自旋相互作用 为了确定内源性位点之间的距离， 顺磁中心 第二个是使用测量的 的自旋晶格弛豫速率的温度依赖性的稳定的 酪氨酸自由基的光系统II探测的磁性 Mn 4团簇。这些实验将为利用电子 确定电子距离的自旋-晶格弛豫测量 金属离子团簇在其他氧化还原体系中的迁移和磁性 蛋白质复合物更好地理解电子的机制 为了应用EPR，蛋白质中的自旋弛豫也是必不可少的 光谱学来研究电子转移蛋白质的动力学， 脉冲EPR在金属蛋白中的应用

Energy transduction in respiration and photosynthesis involves the coupling of electron-transfer reactions to the generation of an electrochemical gradient across a bilayer membrane. Although the sequence and kinetics of the electron-transfer reactions are by and large known in these systems, much less is known about the spatial arrangement of the electron carriers and the distances between them. Ideally, the electron carriers could be used as probes in order to unravel their spatial organization and the distances of long-range electron transfer in the biological electron-transport processes. The long-term goals of this project are to use electron spin-relaxation enhancement measurements to determine distances between redox-active sites in proteins and to probe the magnetic properties of multinuclear metal ion clusters. The first objective is to characterize the effects of pairwise magnetic interactions on the electron spin-relaxation rates in samples in which the distance between interacting centers and the magnetic properties of each center are known. In this study, the reaction center protein from Rb. sphaeroides will be used as a model system. This protein can be prepared in a state that contains two paramagnetic sites at a fixed distance of 28 Angstroms. The electron spin-relaxation rate enhancement of the bacteriochlorophyll special pair cation radical due to a magnetic interaction with the nonheme Fe2+, or Mn2+ or Cu2+ in the Fe2+ site, will be systematically investigated by varying the metal ions and the temperature in both non- oriented and oriented samples. These data will be used to determine the limits of applicability of, and to provide data from which to extend, the theories for spin-relaxation enhancement. The second objective is to use measurements of electron spin-lattice relaxation rate enhancements to determine the spatial organization and magnetic properties of redox sites in cytochrome c oxidase, photosystem II, and nitric oxide synthase, three systems for which structural information is not available. Two types of applications will be pursued in order to obtain structural and magnetic information in these three systems and also to establish the general applicability of this method to determine distances and magnetic data in other systems. The first application is to measure spin-spin interactions between endogenous sites in order to determine the distances between paramagnetic centers. The second is to use measurements of the temperature dependence of the spin-lattice relaxation rate of the stable tyrosine radical in photosystem II to probe the magnetic properties of the Mn4 cluster. These experiments will provide a basis for using electron spin-lattice relaxation measurements to determine distances of electron transfer and magnetic properties of metal ion clusters in other redox protein complexes. A better understanding of the mechanisms of electron spin relaxation in proteins is also essential in order to apply EPR spectroscopy to study dynamics in electron-transfer proteins and for applications of pulsed EPR to metalloproteins.