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.

泛醇：细胞色素c氧化还原酶（bc 1复合物）是一种重要的 细胞能量转导的组成部分，并参与形成 产生ATP所必需的膜电位和质子梯度。 这个项目的长期目标是了解，在详细的分子 术语，cytbc 1复合物在电子转移过程中如何起作用， 矢量质子移位 这种广泛的复杂性对于 活细胞，其不正常的功能导致严重的神经和 肌肉疾病。 我们的实验系统来自于原核生物 荚膜红杆菌是线粒体和线粒体膜的模式生物， 叶绿体能量传递 以前， 利用分子遗传学技术建立了Cytbc 1复合体。 的 其活性结构域之一的位置，即醌醇氧化（Qo）位点，是 现在从抑制剂抗性（InhR）突变体的分析中出现。 这 该项目将继续对细胞色素T进行分子遗传学和生物化学分析 bc 1复合物，以关联其功能位点[醌醇氧化（Qz，o，p）] 和醌还原（Qc，i，n）]与其结构的不同区域 亚基（FeS蛋白、cyt B、cyt c1）。 为此，（1）联合国的作用 位于Qo附近的cyt B的特定氨基酸残基 结构域将通过饱和诱变进行分析;（2）亚基内和亚基内突变， 将由第二个研究中心分析Qo结构域的亚基间相互作用 非功能性cyt B突变体的抑制子;（3） 将FeS蛋白的普遍保守残基连接到Qo位点， 通过诱变评估;（4）细菌bc 1的Qi结构域的定位 将通过分析Rhodocellum的InhR突变体来定义复合物 rubrum，一个自然敏感的物种，并通过构建Qi突变体， R. capsulatus;和（5）结构和功能的影响， 在cyt bH和bL血红素的轴向配体之间添加残基 会得到考验 这些突变体的特征是遗传， 生理生化分析。 从这种细菌中获得的见解 系统通常适用于结构更复杂，但 功能相似的线粒体和叶绿体氧化还原酶， 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到 膜蛋白亚基和它们的假体之间的相互作用 在它们的生物发生和组装过程中。