MOLECULAR GENETICS OF THE CYTOCHROME BC1 C COMPLEX

细胞色素 BC1 C 复合物的分子遗传学

• 批准号: 2179225
• 负责人: M. FEVZI DALDAL
• 金额: $ 20.83万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-12-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179225
• 关键词: Rhodospirillales; Rhodospirillum; bacterial genetics; bioenergetics; chloroplasts; cytochrome c; cytochrome oxidase; electron transport; enzyme complex; enzyme structure; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic recombination; genetic strain; membrane potentials; membrane proteins; mitochondria; molecular cloning; mutant; nucleic acid sequence; oxidation; oxidoreductase inhibitor; photosynthetic bacteria; protein structure function; quinones; spectrometry

The ubiquinol:cytochrome c oxidoreductase (bc1 complex) is an essential component of cellular energy transduction, and is involved in the formation of a membrane potential and a proton gradient necessary to produce ATP. The long term goal of this project is to understand, in detailed molecular terms, how the cyt bc1 complex functions during electron transfer and vectorial proton translocation. This widespread complex is essential for living cells, and its improper function leads to severe neurological and muscular diseases. Our experimental system derives from the procaryote Rhodobacter capsulatus which is a model organism for mitochondrial and chloroplast energy transduction. Previously, the primary structure of the cyt bc1 complex was established using molecular genetic techniques. The location of one of its active domains, the quinol oxidation (Qo) site, is now emerging from the analysis of inhibitor resistant (InhR) mutants. This project will continue molecular genetic and biochemical analyses of the cyt bc1 complex to correlate its functional sites [quinol oxidation (Qz,o,p) and quinone reduction (Qc,i,n)] with different regions of its structural subunits (FeS protein, cyt b, cyt c1). For this purpose, (1) Role of the specific amino acid residues of cyt b located in the vicinity of the Qo domain will be analyzed by saturation mutagenesis; (2) Intrasubunit and intersubunit interactions at the Qo domain will be analyzed by second site suppressors of non functional cyt b mutants; (3) Contribution of the universally conserved residues of the FeS protein to the Qo site will be assessed by mutagenesis; (4) Location of the Qi domain of a bacterial bc1 complex will be defined by analysis of InhR mutants of Rhodospirillum rubrum, a naturally sensitive species and by construction of Qi mutants of R. capsulatus; and (5) Structural and functional implications of the addition of a residue between the axial ligands of the cyt bH and bL hemes will be tested. These mutants will be characterized by genetic, physiological and biochemical analyses. Insights gained in this bacterial system are generally applicable to the structurally more complex and yet functionally similar mitochondrial and chloroplast oxidoreductases, and are relevant to the understanding of the molecular basis of mitochondrial diseases. This work also contributes to a better recognition of the interactions between the subunits of membrane proteins and their prosthetic groups during their biogenesis and assembly.

泛素醇：细胞色素C氧化还原酶（BC1络合物）是必不可少的 细胞能量转导的成分，并参与形成 产生ATP所需的膜电位和质子梯度。 该项目的长期目标是详细地了解 术语，Cyt BC1复合物在电子传输过程中的功能以及 矢量质子易位。 这个广泛的综合体对于 活细胞及其功能不当会导致严重的神经系统和 肌肉疾病。 我们的实验系统源自procaryote Rhodobacter capsulatus是线粒体和 叶绿体能量转导。 以前，主要结构 使用分子遗传技术建立了CYT BC1复合物。 这 其活性域之一的位置，喹诺尔氧化（QO）位置为 现在从抑制剂（INHR）突变体的分析中出现。 这 项目将继续进行CYT的分子遗传和生化分析 BC1复合物将其功能位点相关[Quinol氧化（QZ，O，P） 和喹酮还原（QC，i，n）]具有不同的结构区域 亚基（FES蛋白，Cyt B，Cyt C1）。 为此，（1） 位于QO附近的Cyt B的特异性氨基酸残基 结构域将通过饱和诱变进行分析。 （2）亚基内和 QO域的sububunit相互作用将通过第二站点分析 非功能性Cyt B突变体的抑制剂； （3） FES蛋白的普遍保守残基将是Qo位点的 通过诱变评估； （4）细菌BC1的Qi域的位置 复合物将通过分析杜鹃花的INHR突变体的分析来定义 rubrum，一种天然敏感的物种，并通过构造Qi突变体 R. capsulatus; （5） 在Cyt BH和Bl Hemes的轴向配体之间添加残留物 将进行测试。 这些突变体将以遗传为特征 生理和生化分析。 在此细菌中获得的见解 系统通常适用于结构上更复杂的系统 在功能上相似的线粒体和叶绿体氧化还原酶，并且是 与理解线粒体的分子基础有关 疾病。 这项工作也有助于更好地认识 膜蛋白的亚基及其假肢之间的相互作用 小组在其生物发生和组装过程中。