MOLECULAR ARCHITECTURE OF UQH2: CYT C2 OXIDOREDUCTASE

在过去的几年里，电子转移链的机制 产生用于偶联到ATP合成的质子梯度， 为若干系统大致建立了一个框架。 对苯二酚氧化 呼吸和光合作用复合体都通过Q循环起作用 机制，并催化氧化的醌醇，还原两个 相当于细胞色素c（或相当于蛋白质），转移两个 H+/2 e-穿过膜，并释放两个额外的质子， 水相含有细胞色素C。 我们建议使用一种组合 的技术来研究的三个催化位点的 泛醇：Rps的细胞色素c2氧化还原酶。sphaeroides 在这 光合细菌，复合物的翻转可以很容易地启动 动力学和热力学方法已经被 用来详细说明这个装置 述催化位点 确定的是泛醇氧化酶位点，醌（半醌）还原酶 位点和细胞色素C2还原酶位点。 类似的动力学方法可以 用于研究其中催化位点已被修饰的络合物， 突变 通过研究病变所在的突变株， 在一个特定的催化位点，我们建议将肽 对特定位点有贡献的序列。 然后我们将使用网站指导 诱变以特异性修饰催化位点的氨基酸， 并分析其对催化作用的影响。 我们将补充这些 研究与调查的地形复杂的在 膜，使用针对特定亲水性片段的抗体 的多肽链。 我们希望能够绘制出详细的 催化位点的结构和拓扑结构，并显示如何具体 基团有助于催化机制。

During the past few years, the mechanisms by which electron transfer chains generate proton gradients for coupling to ATP synthesis have been established in broad outline for a number of systems. The quinol oxidizing complexes of respiration and photosynthesis all act through Q-cycle mechanisms, and catalyse oxidation of a quinol, reduction of two equivalents of cytochrome c (or an equivalent protein), the transfer of two H+/2e- across the membrane, and the release of two additional protons to the aqueous phase containing cytochrome c. We propose to use a combination of techniques to study the three catalytic sites of the ubiquinol:cytochrome c2 oxidoreductase of Rps. sphaeroides. In this photosynthetic bacterium, turn-over of the complex can be easily initiated by flashes of light, and kinetic and thermodynamic approaches have been used to characterise the mechanism in some detail. The catalytic sites identified are a ubiquinol oxidase site, a quinone (semiquinone) reductase site, and a cytochrome c2 reductase site. Similar kinetic methods can be used to study the complex in which catalytic sites have been modified by mutation. By studying mutant strains in which the lesion has been located at a specific catalytic site, we propose to characterise the peptide sequences contributing to particular sites. We will then use site directed mutagenesis to specifically modify the amino acids at the catalytic site, and analyse the consequent effects on catalysis. We will complement these studies with an investigation of the topography of the complex in the membrane, using antibodies directed against specific hydrophilic segments of the polypeptide chain. We expect to be able to map the detailed architecture and topology of the catalytic sites, and to show how specific groups contribute to the catalytic mechanism.