STRUCTURE/FUNCTION OF THE CYTOCHROME B6F COMPLEX

细胞色素 B6F 复合物的结构/功能

• 批准号: 6208535
• 负责人: William A. Cramer
• 金额: $ 24.02万
• 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/6208535
• 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b6 f复合物与线粒体呼吸链的细胞色素bc 1复合物具有许多结构-功能同源性。 这主要是由于细胞色素B的特性，特别是在膜的p侧上的标记。 从细胞色素f和Rieske（ISP）蛋白的高分辨率结构中，已知bc 1和b6 f之间的外周结构域显著不同。 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，并已从它们的进化起源，和域的ISP远端膜折叠不同的b6 f和bc 1复合物。 然而，ISP域含有铁硫簇，必须接近膜有一个保守的折叠。 因此，关于b6 f复合体结构-功能的详细信息补充了bc 1的结构-功能，并且似乎可能提供详细功能机制的差异。 一种来自嗜热蓝细菌M. laminosus，会提供这些信息。 晶体目前显示出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，结构的解将由于我们具有复合物的p-侧、细胞色素f和铁-硫蛋白（复合物总质量的40%）的高分辨率结构（小于2.0埃单位）的事实而加速。 由结构决定的功能问题包括正侧醌的位置和功能、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，ISP的局部移动性将在体内、原位和体外进行分析。 将研究高势链和低势链的非协调还原的基础。在Rieske蛋白中的保守芳香族化合物对电子转移的催化作用，以及在cytf中它们屏蔽血红素的地方，将由我的诱变来测试。 水链在蛋白质内电子和质子转移耦合中的作用将通过D2 O中的停流动力学来研究，同时通过FTIR来研究cytf中结合H2O的性质。 与钌衍生物的cytf，“光-cytf”，光诱导的蛋白质内电子转移速率，最佳路径的蛋白质内电子转移，和重组能量将被测量。 钌化的细胞因子也将用于研究蛋白质内质子化-去质子化在特定的羧酸，与耦合电子和质子转移。