STRUCTURE/FUNCTION OF THE CYTOCHROME B6F COMPLEX

细胞色素 B6F 复合物的结构/功能

• 批准号: 2909266
• 负责人: William A. Cramer
• 金额: $ 10.07万
• 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/2909266
• 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。这在很大程度上源于细胞色素b的同一性，特别是在膜的p侧。从细胞色素f和Rieske （ISP）蛋白的高分辨率结构中，我们知道bc1和b6f的外周结构域存在显著差异。结构域离膜越远，差异越大。因此，细胞色素f和c1是完全不同的蛋白质，它们的进化起源不同，并且在b6f和bc1复合体中，远端膜的ISP结构域折叠方式不同。然而，含有铁硫簇的ISP结构域必须靠近膜，具有保守折叠。因此，关于b6f复合体的结构-功能的详细信息获得了b6f复合体的结构-功能，似乎可能提供了详细功能机制的差异。一种高分辨率的3-D晶体结构的b6f复合体，来自嗜热嗜冷细菌M. laminosus，将提供这一信息。晶体目前表现出10埃单位的有序衍射。当获得适合于结构分析的衍射（小于或等于3埃单位）时，由于我们对配合物的p侧，细胞色素f和铁硫蛋白（占配合物总质量的40%）具有高分辨率结构（小于2.0埃单位），因此结构的解决将加快。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。从现有的p侧结构出发，分析了ISP在体内、原位和体外的局部迁移率。研究了高、低电位链非协调还原的基础。Rieske蛋白和cytf中保护血红素的保守芳香对电子转移的催化作用将被测试为诱变。水链在蛋白质内电子和质子转移耦合中的作用将通过D2O中的停止流动动力学来检验，并通过FTIR来检验cytf中结合的H2O的性质。利用cytf的钌衍生物“photocytf”，光诱导的蛋白内电子转移速率、蛋白内电子转移的最佳路径和重组能将被测量。钌化cytf也将用于研究特定羧酸的蛋白内质子化-去质子化，与耦合电子和质子转移有关。