ELECTRON-TRANSFER PROTEINS

电子传递蛋白

• 批准号: 2173590
• 负责人: FRANCIS S MILLETT
• 金额: $ 25.19万
• 依托单位: UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-06-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2173590
• 关键词: Rhodopseudomonas; X ray crystallography; bioenergetics; cytochrome b; cytochrome b5 reductase; cytochrome c; cytochrome oxidase; electron transport; enzyme complex; enzyme mechanism; hemoprotein structure; high performance liquid chromatography; hydrogen channel; iron sulfur protein; laser spectrometry; membrane transport proteins; metalloenzyme; oxidation reduction reaction; peroxidases; protein reconstitution; protein sequence; protein structure function; ruthenium; site directed mutagenesis

The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions to numerous biological processes, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. We have recently introduced a new method to study biological electron transfer that utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(ll)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of inter protein electron transfer. Over twenty singly labeled derivatives of cytochrome c have now been prepared and characterized. One of the most remarkable properties of Ru(ll) is that it can be photoexcited to a metal-to-ligand charge-transfer state, Ru(lI*), which is a strong reducing agent and can rapidly transfer an electron to the heme group Fe(llI) in cytochrome c. Rate constants up to 3 x 10(7) s-1 are observed for derivatives with separations of about 12 Angstroms between the ruthenium and heme groups. We are using this new technique to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome c1, cytochrome c peroxidase. The rate constants for these reactions range from 10(4) to over 10(6) s-1 , and are up to three orders of magnitude larger than previous estimates. The specific aims for the next grant period are to: 1) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c peroxidase that brings together rapid kinetics, site-directed mutagenesis, and X-ray crystallography. The rate constant, reorganization energy, interaction domain, and pathway of each electron transfer step in the mechanism will be determined. 2) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c oxidase. A major goal will be to determine the pathway and kinetics of electron transfer from cytochrome c through Cu-a and heme a to the heme a3--Cu-B binuclear center under coupled turnover conditions. 3) Carry out a detailed study of the electron transfer reaction between cytochrome c and the cytochrome bc1 complex. A major goal will be to determine the pathway and kinetics of electron transfer from the Rieske iron-sulfur center to cytochrome c1 and to cytochrome c.

NIH GM20488支持的研究计划的远程目标是 开发和应用新技术来研究生物电子转移 反应。尽管这些反应对众多反应很重要 生物过程，相对较少的技术可以测量 在A中的两个氧化还原中心之间的电子转移速率 蛋白质复合物。我们最近引入了一种研究的新方法 利用共价附加的生物电子转移 Tris（Bipyridine）ruthenium group [ru（ll）]。几种策略已经 开发用于设计和合成钌标记的氧化还原蛋白 用于测量蛋白质电子转移的优化。 超过二十个单一标记的细胞色素C衍生物已成为 准备和表征。最杰出的特性之一 ru（ll）是可以将其光电在金属到水平的电荷转移到转移 状态，ru（li*），它是强大的还原剂，可以快速转移 在细胞色素c中的血红素组Fe（LLI）的电子c。速率常数 对于分离约12的衍生物，观察到3 x 10（7）S-1 唯一和血红素组之间的埃埃斯特罗姆。我们正在使用这个新的 测量细胞色素c之间的跨复合电子转移的技术 及其生理伴侣，细胞色素C氧化酶，细胞色素C1， 细胞色素C过氧化物酶。这些反应的速率常数范围从 10（4）至10（6）S-1，最多三个数量级 比以前的估计值。下一个赠款期的具体目标是 至：1）对电子转移反应进行详细研究 在细胞色素C和细胞色素C过氧化物酶之间，将其融合在一起 快速动力学，定向诱变和X射线晶体学。这 速率常数，重组能量，相互作用域和途径 将确定机制中的每个电子传输步骤。 2）随身携带 详细研究细胞色素之间的电子转移反应 C和细胞色素C氧化酶。一个主要目标是确定路径 电子从细胞色素c通过Cu-A和血红素的动力学转移 在耦合周转条件下，a到达血红素A3-CU-B双核中心。 3）对电子转移反应进行详细研究 细胞色素C和细胞色素BC1复合物。一个主要目标是 确定从Rieske的电子传递的途径和动力学 铁硫心中心至细胞色素C1和细胞色素c。