DISTANCE MEASUREMENTS IN METALLOPROTEINS

金属蛋白质的距离测量

• 批准号: 2178370
• 负责人: GARY W BRUDVIG
• 金额: $ 16.85万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-23 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178370
• 关键词: DISTANCE; MEASUREMENTS; METALLOPROTEINS

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.

呼吸和光合作用的能量转导涉及 电子转移反应与生成的耦合 跨双层膜的电化学梯度。虽然序列 电子转移反应的动力学在很大程度上已知 这些系统，对有关的空间布置的了解少得多 电子载体及其之间的距离。理想情况下，电子 载体可以用作探针以揭示其空间 组织和远程电子转移的距离 生物电子传输过程。这个长期目标 项目将使用电子自旋 - 浮肿增强测量值 确定蛋白质中的氧化还原活性位点与探测 多核金属离子簇的磁性。第一个 目的是表征成对磁相互作用的影响 在距离的样品中的电子自旋 - 浮雕速率上 在相互作用的中心和每个中心的磁性之间 已知。在这项研究中，反应中心蛋白来自RB。 sphaeroides 将用作模型系统。该蛋白可以在状态下制备 其中包含两个固定距离为28埃的磁磁性位点。 电子自旋 - - 闪烁率提高了细菌氯苯酚 由于与非血红素的磁相互作用，特殊对阳离子阳离子自由基 Fe2+站点中的Fe2+或Mn2+或Cu2+将系统地为 通过改变金属离子和两个非 - 定向和定向样品。这些数据将用于确定 适用性的限制，并提供可以扩展的数据， 自旋 - 闪烁增强的理论。第二个目标是使用 电子自旋晶格松弛率的测量提高到 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，三个 无法提供结构信息的系统。两种类型 为了获得结构和磁性，将进行应用 这三个系统中的信息，也建立一般 此方法的适用性来确定距离和磁数据 其他系统。第一个应用是测量自旋旋转相互作用 内源地点之间以确定 顺磁心中心。 第二个是使用 稳定的自旋晶格松弛率的温度依赖性 光系统II中的酪氨酸自由基探测 MN4群集。这些实验将为使用电子提供基础 自旋晶格松弛测量值以确定电子的距离 其他氧化还原中金属离子簇的传递和磁性 蛋白质复合物。更好地理解电子机制 蛋白质中的自旋松弛对于应用EPR也是必不可少的 研究电子转移蛋白和用于研究动力学的光谱和用于 脉冲EPR应用于金属蛋白。