BIOENERGETICS OF THE CYTOCHROME BC1 COMPLEX

描述：本研究项目的总体目标是调查 细胞色素B及其铁硫蛋白ISP的作用 bc 1复合物在电子转移和质子释放反应中的作用， 发生在复合物中的喹啉氧化位点。 的重要性 加强了对线粒体生物能量学的深入理解 越来越多的证据表明，电子转移链中的缺陷可能 导致人类退化性疾病和衰老过程。 具体来说，我们将研究质子泵送的机制。 和电子转移似乎是解耦使用酵母细胞色素B 保留显著的电子转移活性但不能生长的突变体 呼吸基质上。 质子泵和电子转移活性 将在线粒体和从野生型分离的bc 1复合物中进行测定 酵母和来自含有位于附近或附近氨基酸突变的菌株 在细胞色素B螺旋CD中。NCD-4的荧光特性， 与位于酵母螺旋cd中的天冬氨酸-160结合的DCCD类似物 细胞色素B，将被用来探测可能的变化，在cd螺旋在 突变体中质子泵和电子转移似乎是 分离 酸性氨基酸存在于 细胞色素B的膜外但疏水的螺旋cd参与 质子泵将通过酸性氨基酸的定点突变来检查 类球红球藻螺旋cd中的氨基酸。 ISP定位于细胞色素bc 1 复合物也参与泛醇的氧化。 定点 位于ISP膜外区域的氨基酸的诱变 肯定会. 以确定带电荷的氨基酸残基是否 将ISP有效组装到bc 1复合体中以及 酶活性

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.