STRUCTURE AND FUNCTION OF CUA IN CYTOCHROME C OXIDASE

细胞色素C氧化酶催化氧的4电子还原为水 所有高等生命形式中细胞呼吸的最后一步。在 在发现50年后，人们已经发现该酶含有4个 氧化还原活性金属中心。最近，情况变得很明显， 与这种氧化还原化学作用相结合，细胞色素c氧化酶也能泵出质子。 穿过线粒体膜以增加电化学能量 由呼吸作用产生的梯度。这种氧化还原相关的机制 泵浦仍然难以捉摸，目前仍不清楚哪个金属中心，如果 任何，都是牵涉其中的。一段时间前有人提出，从光谱角度看 不同寻常的铜(A)中心具有前所未有的二硫键配位， 这种协调对于它在质子泵中的作用至关重要。上一首 研究结果表明，铜(A)至少含有一种半胱氨酸 配体，但不寻常的第二个半胱氨酸配体的提议仍然存在 未经证实。进化信息还要求铜(A)是 由接近的两种特定半胱氨酸中的一种或两种配位 另一种在亚基II的蛋白质序列高度保守的区域。 直到最近，真核细胞色素c的定点突变 氧化物酶在技术上是不可能的。跨膜酶含有 2个血红素铁蛋白，由12个或更多不同亚基组成，3个亚基编码 线粒体DNA(其中一个或多个包含所有4个金属中心)，以及 其余的由核基因编码。然而，它最近变成了 有可能用外源DNA转化酵母线粒体， 幸运的是，这个程序是使用亚基的基因(COX2)开发的 酵母菌细胞色素C氧化酶。 这项提议的目标是开发一种探测金属的新方法。 以结构和功能为中心，测试和进一步细化详细模型 协调铜(A)并评估该中心的参与 在电子转移和质子泵浦的机制中。个人赛斯， 亚基II中的His和Tyr残基与 上述模型将通过酵母中的定点突变而改变。这个 这些突变对铜(A)位结构和功能的影响 在酵母体内和在洗涤剂中溶解的酶将通过 功能分析以及光学和EPR光谱分析。如果这种酶 未能正确组装给定的突变、第二位点回复突变 将被选中，所产生的突变酶将类似地 分析过了。这些结果将与世界银行提供的预测进行比较 结构模型。最后，研究了这些突变体的质子泵活性。 将进行比较，以提供支持或反对模型的有力证据 由这个不同寻常的铜中心泵送的氧化还原关联的质子。

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.