Studies of the Cyanide-resistant Alternative Oxidase

植物线粒体除了细胞色素c氧化酶途径外，还具有一条抗氰化物的替代氧化酶途径。在呼吸过程中，替代氧化酶将氧气还原为水，但将产生的能量以热的形式释放出来，而不是储存起来。该途径在植物中广泛存在，促使人们探索替代氧化酶在植物新陈代谢中的作用。植物酶是二聚体，由两个相同的亚基组成，如果亚基通过二硫键连接则失活。如果这种调节键被减少，那么这种酶就可以被小的代谢物a-酮酸完全激活。真菌线粒体中也存在一种替代的氧化酶，但它是单体的，由嘌呤核苷酸而不是α-酮酸激活。该项目的一个目标是确定植物和真菌蛋白的哪些区域负责它们不同的结构和调节功能。为了实现这一目标，将使用重组DNA技术创建嵌合蛋白，并检测其性质。该项目的第二个目标是解决在分离的线粒体中展示的调节特性是否对体内替代氧化酶的功能重要。在一种方法中，将观察在实验条件下孤立的膜系统中蛋白质的调节二硫键的行为，并将其与其在更复杂的环境(线粒体、细胞和叶子)中的行为进行比较。第二种方法将使用转基因拟南芥植物，这些植物要么表达很高或很低水平的正常替代氧化酶，要么表达高水平的永久激活的突变替代氧化酶。将检查这些植物对各种环境变量的反应，以确定在哪些条件下存在或不存在替代氧化酶及其调节功能是有益的或有害的。这一结果应该会增加对调节功能对其在体内功能的重要性以及替代氧化酶参与植物对环境反应的理解。由于替代氧化酶浪费了线粒体中的能量，线粒体是细胞中的主要能量产生者，人们曾认为这种酶的活性可能会导致植物生产力下降。然而，越来越多的证据表明，氧化酶可能反而对植物在广泛的生物和非生物胁迫下生存的能力起着重要作用。这里描述的项目将通过研究植物新陈代谢中替代氧化酶调节的性质和作用来增加这方面的知识。

Plant mitochondria possess a cyanide-resistant alternative oxidase pathway in addition to the cytochrome c oxidase pathway. During respiration, the alternative oxidase reduces oxygen to water, but releases the generated energy as heat, rather than storing it. The pathway is widespread among plants, leading to a search for the role of the alternative oxidase in plant metabolism. The plant enzyme is dimeric, consisting of two identical subunits, and is inactivated if the subunits are united by a disulfide bond. If this regulatory bond is reduced, the enzyme can then be fully activated by small metabolites, a-keto acids. An alternative oxidase is also present in fungal mitochondria, but is monomeric and is activated by purine nucleotides rather than alpha-keto acids. One goal of this project is to determine which regions of the plant and fungal proteins are responsible for their different structural and regulatory features. To accomplish this, chimeric proteins will be created using recombinant DNA technology and their properties examined. The second goal of the project is to address whether the regulatory properties demonstrated in isolated mitochondria are important for the function of the alternative oxidase in vivo. In one approach, the behavior of the protein's regulatory disulfide bond in an isolated membrane system under experimental conditions will be observed and compared to its behavior in more complex environments (mitochondria, cells, and leaves). A second approach will use transgenic Arabidopsis plants that express either very high or low levels of normal alternative oxidase, or high levels of a mutated alternative oxidase that is permanently activated. These plants will be examined for their responses to a variety of environmental variables to determine conditions under which the presence or absence of the alternative oxidase and its regulatory features is beneficial or detrimental. The outcome should increase understanding of how important the regulatory features are to its function in vivo and of the alternative oxidase's involvement in plant responses to the environment. Because the alternative oxidase wastes energy in mitochondria, which are major energy-producers in the cell, it had been thought that this enzyme's activity could lead to reduced plant productivity. Increasing evidence, however, indicates that the oxidase may instead be important for the plant's ability to survive a wide range of biotic and abiotic stresses. The project described here will add to this knowledge through its investigation of the nature and role of alternative oxidase regulation in plant metabolism.