STRUCTURE/FUNCTION OF THE CYTOCHROME B6F COMPLEX

细胞色素 B6F 复合物的结构/功能

• 批准号: 6476484
• 负责人: William A. Cramer
• 金额: $ 25.22万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476484
• 关键词: X ray crystallography; crystallization; cytochrome b; cytochromes; electron transport; enzyme complex; iron sulfur protein; laboratory rabbit; membrane proteins; oxidation reduction reaction; photochemistry; photosynthetic bacteria; protein structure function; protonation; ruthenium; stop flow technique

The cytochrome b6f complex has many structure-function homologies with the cytochrome bc1 complex of the mitochondrial respiratory chain. Much of this arises from the identity of cytochrome b, especially marked on the p-side of the membrane. From the high resolution structures of cytochrome f and the Rieske (ISP) protein, it is known that the peripheral domains differ significantly between bc1 and b6f. The further the domain from the membrane, the greater the difference. Thus, cytochromes f and c1 are completely different proteins and have been from their evolutionary origin, and the domain of the ISP distal to membrane folds differently in the b6f and bc1 complexes. However, the ISP domain containing the iron-sulfur cluster that must come close to the membrane has a conserved fold. Thus, detailed information on structure-function for the b6f complex complements that obtained for the bc1 and seems likely to provide differences in detailed functional mechanisms. A high resolution structure of 3-D crystals of the b6f complex from the thermophilic cy- anobacterium, M. laminosus, would provide this information. The crystals presently show ordered diffraction to 10 Angstrom units. When diffraction (less than or equal to 3 Angstrom units) appropriate for a structure analysis is obtained, the solution of the structure will be expedited by the fact that we have high resolution structures (less than 2.0 Angstrom units) for the p- side of the complex, cytochrome f and the iron-sulfur protein, 40 percent of the total mass of the complex. Issues of function to be determined by the structure include the position and function of the n-side quinone, the pathway of trans-membrane H+ transfer, and the role of intramembrane bound water. From the existing p- side structures, the local mobility of the ISP will be analyzed in vivo, in situ, and in vitro. The basis for the non-concerted reduction of high and low potential chains will be studied. Catalysis of electron transfer by conserved aromats in the Rieske protein, and in cytf where they shield the heme, will be tested my mutagenesis. The role of the water chain in the coupling of intraprotein electron and proton transfer will be examined by stopped flow kinetics in D2O, together with the properties of the bound H2O in cytf by FTIR. With a ruthenium derivative of cytf, "photo-cytf", light-induced intraprotein electron transfer rates, optimum paths of intraprotein electron transfer, and reorganization energy will be measured. Ruthenated cytf will also be used to investigate intraprotein protonation- deprotonation at specific carboxylates, associated with coupled electron and proton transfer.

细胞色素 b6f 复合物与线粒体呼吸链的细胞色素 bc1 复合物具有许多结构功能同源性。 这很大程度上源于细胞色素 b 的特性，特别是在膜的 p 侧标记。 从细胞色素 f 和 Rieske (ISP) 蛋白的高分辨率结构可知，bc1 和 b6f 的外围结构域存在显着差异。 域距膜越远，差异越大。 因此，细胞色素 f 和 c1 是完全不同的蛋白质，并且具有其进化起源，并且膜远端的 ISP 结构域在 b6f 和 bc1 复合物中的折叠方式不同。 然而，包含必须靠近膜的铁硫簇的 ISP 结构域具有保守的折叠。 因此，关于 b6f 复合体结构功能的详细信息补充了 bc1 获得的信息，并且似乎可能提供详细功能机制的差异。 来自嗜热蓝藻 M. laminosus 的 b6f 复合物的 3D 晶体的高分辨率结构将提供这一信息。 目前晶体显示出 10 埃单位的有序衍射。当获得适合结构分析的衍射（小于或等于 3 埃单位）时，由于我们对复合物的 p 侧、细胞色素 f 和铁硫蛋白（占复合物总质量的 40%）具有高分辨率结构（小于 2.0 埃单位），因此将加快结构的解析。 由结构决定的功能问题包括n侧醌的位置和功能、跨膜H+转移的途径以及膜内结合水的作用。 根据现有的 p 侧结构，将在体内、原位和体外分析 ISP 的局部迁移率。 将研究高低电势链非协同还原的基础。 Rieske 蛋白中的保守芳香族以及保护血红素的 cytf 中的保守芳香族对电子转移的催化作用将被测试我的诱变。 水链在蛋白质内电子和质子转移耦合中的作用将通过 D2O 中的停流动力学以及 FTIR 中 cytf 中结合 H2O 的性质进行检查。 使用cytf的钌衍生物“光细胞色素”，将测量光诱导的蛋白质内电子转移速率、蛋白质内电子转移的最佳路径和重组能。 钌酸 cytf 还将用于研究特定羧酸盐处的蛋白内质子化-去质子化，与耦合电子和质子转移相关。