STRUCTURE AND FUNCTION OF CUA IN CYTOCHROME C OXIDASE

细胞色素c氧化酶催化O2的4电子还原为水， 所有高等生物细胞呼吸的最后一步 在 发现50年来，人们发现这种酶含有4 氧化还原活性金属中心。 最近，人们清楚地看到， 与这种氧化还原化学反应相结合，细胞色素c氧化酶也将质子 穿过线粒体膜增加电化学能量 呼吸产生的梯度。 这种与氧化还原有关的机制 泵浦仍然难以捉摸，仍然不知道哪个金属中心，如果 任何人都参与其中。 不久前有人提出， 不寻常的Cu（A）中心具有前所未有的二硫代硫酸盐配位， 这种协调对其在质子泵中的作用至关重要。 先前 研究已经确定地表明Cu（A）具有至少一个半胱氨酸， 配体，但一个不寻常的第二个半胱氨酸配体的建议仍然存在 未经证实 进化信息还要求Cu（A）是 由两个特定的半胱氨酸中的一个或两个协调， 另一个在亚基II的蛋白质序列的高度保守区域中。 直到最近，真核细胞色素c的定点突变 氧化酶在技术上是不可能的。 跨膜酶含有 2个血红素铁，由12个或更多个不同的亚基组成，3个由 线粒体DNA（其中一个或多个含有所有4个金属中心），以及 其余的由核基因编码。 然而，最近却成为 可以用外源DNA转化酵母线粒体， 幸运的是，该程序是使用亚基基因（COX2）开发的， 酵母细胞色素c氧化酶。 该提案的目标是开发一种探测金属的新方法 中心的结构和功能，以测试和进一步完善详细的模型 为协调铜（A），并评估该中心的参与 电子转移和质子泵的机制。 单个Cys， 亚基II内的His和Tyr残基，其已经参与了 上述模型将通过酵母中的定点诱变来改变。 的 这些突变对Cu（A）位点的结构和功能的影响 在酵母体内和在洗涤剂溶解酶中，将通过 功能测定以及光学和EPR光谱。 如果酶 无法正确组装特定突变，第二位点回复突变体 将被选择，并且所得的突变酶将类似地 分析了 这些结果将与预测提供的 结构模型 最后，这些突变体的质子泵活动 将进行比较，以提供强有力的证据支持或反对一个模型 这个不寻常的铜中心产生的与氧化还原有关的质子泵。

Cytochrome c oxidase catalyzes the 4-electron reduction of O2 to water in the final step of cellular respiration in all higher forms of life. In the 50 years since its discovery, it has been found that the enzyme contains 4 redox-active metal centers. More recently, it has become clear that coupled to this redox chemistry, cytochrome c oxidase also pumps protons across the mitochondrial membrane to add to the electrochemical energy gradient generated by respiration. The mechanism of this redox-linked pumping remains elusive and it is still not known which metal center, if any, is involved. It was proposed some time ago that the spectroscopically unusual Cu(A) center has an unprecedented dithiolate coordination and that this coordination is central to its role in proton pumping. Previous studies have conclusively shown that Cu(A) has at least one cysteine ligand, but the proposal of an unusual second cysteine ligand remains unproven. Evolutionary information additionally requires that Cu(A) be coordinated by one or both of two specific cysteines found close to each other in a highly conserved region of the protein sequence for subunit II. Until recently, site-directed mutagenesis of eukaryotic cytochrome c oxidase was not technically possible. The transmembranous enzyme contains 2 heme irons and is composed of 12 or more different subunits, 3 encoded by mitochondrial DNA (one or more of these contains all 4 metal centers), and the remaining encoded by nuclear genes. However, it has recently become possible to transform yeast mitochondria with exogenous DNA and, fortuitously, the procedure was developed using the gene (COX2) for subunit II of yeast cytochrome c oxidase. The goals of this proposal are to develop a new approach to probing metal center structure and function, to test and further refine detailed models for the coordination of Cu(A) and to assess the involvement of this center in the mechanism of electron transfer and proton pumping. Individual Cys, His and Tyr residues within subunit II which have been implicated in the above models will be altered by site-directed mutagenesis in yeast. The effects of these mutations on the structure and function of the Cu(A) site in yeast in vivo and in detergent-solubilized enzyme will be probed by functional assays and by optical and EPR spectroscopies. If the enzyme fails to assemble correctly for a given mutation, second-site revertants will be selected for, and the resultant mutant enzymes will be similarly analyzed. These results will compared with predictions afforded by the structural models. Finally, the proton pumping activities of these mutants will be compared in order to provide strong evidence for or against a model of redox-linked proton pumping by this unusual copper center.