BIOENERGETICS OF THE CYTOCHROME BC1 COMPLEX

细胞色素 BC1 复合物的生物能量学

• 批准号: 2900916
• 负责人: DIANA S. BEATTIE
• 金额: $ 21.79万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900916
• 关键词: Rhodospirillales; SDS polyacrylamide gel electrophoresis; active sites; bioenergetics; cytochrome b; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme mechanism; hydrogen transporting ATP synthase; iron sulfur protein; mitochondria; mutant; protein sequence; site directed mutagenesis; stop flow technique; western blottings; yeasts

DESCRIPTION: The overall goal of this research project is to investigate the role of cytochrome b and the iron-sulfur protein ISP of the cytochrome bc1 complex in the electron transfer and proton releasing reactions which occur at the quinol-oxidizing site in the complex. The importance of an in-depth understanding of mitochondrial bioenergetics has been strengthened by the increasing evidence that defects in the electron transfer chain may contribute to human degenerative diseases and to the aging process. Specifically, we will investigate the mechanism(s) by which proton pumping and electron transfer appear to be uncoupled using yeast cytochrome b mutants which retain significant electron transfer activity but cannot grow on respiratory substrates. Proton pumping and electron transfer activity will be assayed in mitochondria and in bc1 complexes isolated from wild type yeast and from strains containing mutations in amino acids located near or in helix cd of cytochrome b. The fluorescent properties of NCD-4, the analogue of DCCD that binds to aspartate-160 located in helix cd of yeast cytochrome b, will be used to probe possible changes in the cd helix in the mutants in which proton pumping and electron transfer appear to be uncoupled. The suggestion that acidic amino acids localized in extra-membranous, yet hydrophobic, helix cd of cytochrome b are involved in proton pumping will be examined by site-directed mutagenesis of acidic amino acids in helix cd of R sphaeroides. The ISP locate in the cytochrome bc1 complex also participates in the oxidation of ubiquinol. Site directed mutagenesis of amino acids located in extra-membranous regions o the ISP will be. performed to determine whether charged amino acid residues ar required for efficient assembly of the ISP into the bc1 complex and for enzymatic activity.

描述：本研究项目的总体目标是调查 细胞色素b和细胞色素铁硫蛋白的作用 Bc1络合物在电子转移和质子释放反应中 发生在络合物中的苯二酚氧化位置。重要的是一个 对线粒体生物能量学的深入了解得到了加强 由于越来越多的证据表明，电子转移链中的缺陷可能 有助于人类退化性疾病和衰老过程。 具体地说，我们将研究质子泵浦的机制(S) 和电子传递似乎是通过酵母细胞色素b解偶联的。 保持显著电子转移活性但不能生长的突变体 在呼吸基质上。质子泵浦与电子转移活性 将在线粒体和从野生型分离的Bc1复合体中进行检测 酵母菌和含有氨基酸突变的菌株 在细胞色素b的螺旋CD中。NCD-4的荧光性质， 与位于酵母螺旋CD上的天冬氨酸-160结合的DCD类似物 细胞色素b，将被用来探测CD螺旋的可能变化。 其中质子泵浦和电子转移似乎是 不耦合。建议酸性氨基酸定位于 细胞色素b的膜外但疏水性的螺旋cd参与了 质子泵将通过酸性氨基的定点突变进行检测 球藻螺旋体CD中的酸。Isp位于细胞色素Bc1中 络合物还参与泛喹酚的氧化。站点定向 膜外区氨基酸的致突变作用 会是的。执行以确定带电氨基酸残基是否 将isp有效地组装到Bc1复合体中所需的，以及 酶活性。