A GENETIC ANALYSIS FOR THE FUNCTIONAL SITES OF THE

泛醇：细胞色素c氧化还原酶（或bc 1复合物） 参与膜电位和质子的形成 梯度，活细胞的重要过程。 的长期目标 这个项目是为了从分子的角度来理解， bc 1复合物的结构亚基与它们的 相关的假体组来执行这个角色。 作为初始 步骤，bc 1复合物的结构基因， Rhodopterium capsulata是一种光合作用的原核生物， 它们的核苷酸序列已经被分离出来， 测定 该复合物的体内作用也已被证实。 通过构建无效BC 1突变体来评估。 我们现在计划 进行详细的分子遗传分析， bc 1复合物的功能位点（醌醇氧化，醌 还原和细胞色素c还原），其结构 亚基（FeS蛋白、细胞色素B和细胞色素c1）。 为 为此，我们将（1）选择抗特定 BC 1复合物的抑制剂;（2）获得新的BC 1突变体， 组装功能改变的复合物;（3）图谱和序列 这些突变，包括R126的aer 126， 复合物的醌醇氧化位点;（4）改变氨基 酸残基因此被定义为各种其他残基， 定点寡核苷酸诱变， 突变的位点与扰动的功能。 在这方面获得的见解 原核系统在结构上应普遍适用于 更复杂但功能相似的线粒体， 高等生物的叶绿体氧化还原酶，它们通常是 基因分析的结果。 一般来说，这项工作还应 有助于更好地理解功能相互作用 在辅基和结构亚基之间 膜蛋白

The ubiquinol:cytochrome c oxidoreductase (or the bc1 complex) is involved in the formation of the membrane potential and proton gradient, a vital process for living cells. The long term goal of this project is to understand, in molecular terms, how the structural subunits of the bc1 complex interact with their associated prosthetic groups to perform this role. As an initial step, the structural genes of the bc1 complex of Rhodopseudomonas capsulata, a photosynthetic prokaryote, have been isolated and their nucleotide sequences have been determined. The in vivo role of this complex has also been assessed by constructing null bc1 mutants. We now plan to undertake a detailed molecular genetic analysis to correlate the function sites of the bc1 complex (quinol oxidation, quinone reduction, and cytochrome c reduction) with its structural subunits (the FeS protein, cytochrome b, and cytochrome c1). For this purpose we will (1) select mutants resistant to specific inhibitors of the bc1 complex; (2) obtain new bc1 mutants that assemble a functionally altered complex; (3) map and sequence these mutations, including aer126 of R126, a mutant defective in the quinol oxidation site of the complex; and (4) change the amino acid residues thus defined to various other residues by sitedirected oligonucleotide mutagenesis to correlate the mutated sites with the perturbed functions. Insights gained in this prokaryotic system should be generally applicable to structurally more complex but functionally similar mitochondrial and chloroplast oxidoreductases of higher organisms, which are often refractory to genetic analysis. In general, this work should also contribute to a better understanding of functional interactions between the prosthetic groups and the structural subunits of membrane proteins.