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复合体与线粒体呼吸链的细胞色素bc1复合体有许多结构和功能上的同源性。这在很大程度上是由于细胞色素b的身份，特别是在膜的p侧标记的。从细胞色素f和Rieske(Isp)蛋白的高分辨结构可知，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，来自它们的进化起源，并且膜远端的isp结构域在b6f和bc1复合体中折叠不同。然而，含有必须靠近膜的铁-硫簇的ISP域有一个保守的折叠。因此，关于B6f复合体结构功能的详细信息补充了对Bc1的结构-功能信息，并似乎可能在详细的功能机制方面提供差异。来自嗜热细胞厌氧菌的b6f复合体的高分辨率三维晶体结构将提供这一信息。晶体目前显示出10埃的有序衍射单位。当获得适合于结构分析的衍射(小于或等于3埃单位)时，由于我们具有高分辨率结构(小于2.0埃斯特罗姆单位)，即细胞色素f和铁硫蛋白，占复合物总质量的40%，结构的解决将加快。结构决定的功能问题包括n-侧苯二酚的位置和功能，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，将在体内、原位和体外分析isp的局部迁移率。将研究高、低势链非协同还原的基础。Rieske蛋白中保守的芳香族化合物对电子转移的催化作用，以及它们屏蔽血红素的细胞因子，将测试我的诱变作用。水链在蛋白质内电子和质子转移耦合中的作用将通过D2O中的停流动力学和结合的H2O在细胞因子中的FTIR性质来检验。利用细胞因子的一种Ru的衍生物，“光敏细胞因子”，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量。Ruthenated Cytf还将用于研究特定羧基上的蛋白质内质子化-去质子化，与耦合的电子和质子转移有关。