A GENETIC ANALYSIS FOR THE FUNCTIONAL SITES OF THE

泛喹酚：细胞色素c氧化还原酶(或Bc1复合体) 参与了膜电势和质子的形成 梯度，这是活细胞的一个重要过程。的长期目标是 这个项目是用分子的术语来理解 Bc1复合体的结构亚基与它们的 相关的假肢小组来扮演这一角色。作为首字母 STEP，Bc1复合体的结构基因 胶囊红假单胞菌是一种具有光合作用的原核生物。 并对其核苷酸序列进行了分析 下定决心。这种复合体在体内的作用也是 通过构建零bc1突变体进行评估。我们现在计划 进行详细的分子遗传分析，以将 Bc1复合体的功能位点(苯二酚氧化、苯二酚 还原和细胞色素c还原)与其结构 亚基(FeS蛋白、细胞色素b和细胞色素c1)。为 为此，我们将(1)筛选出抗特定病毒的突变体 Bc1复合体的抑制剂；(2)获得新的bc1突变体， 组装一个功能改变的综合体；(3)地图和序列 这些突变，包括R126的Aer126，一个有缺陷的突变 络合物的苯二酚氧化位置；和(4)改变氨基 酸残基如此定义为各种其他残基，由 定点寡核苷酸突变相关基因 功能受到干扰的突变位点。在此过程中获得的见解 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和 高等生物的叶绿体氧化还原酶，通常是 难以进行遗传分析。一般来说，这项工作还应该 有助于更好地理解功能相互作用 在假体基团和结构亚单位之间 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.