Studies of the Plant Cyanide-Resistant Alternative Oxidase

Siedow是植物线粒体的一种不含氰化物的“替代”氧化酶，利用线粒体泛苯二酚池中的电子催化氧还原为水。在这个反应中没有能量守恒，因此交替氧化酶在植物新陈代谢中的作用一直令人费解。最近，在发现线粒体中该酶的活性是如何调节的方面取得了很大进展。交替氧化酶已被证明是二聚体，当两个单体通过二硫键连接时，其活性降低。当这个二硫键对被还原时，酶变得更活跃，并且能够被(-酮酸)刺激。(-酮酸)的作用部位也很可能是在巯基残基上。结合序列分析，对调控机制的研究为了解该酶的重要结构特征提供了线索。另一种酶被预测有一个双铁催化位点，并且已经确定了可能与泛醌底物结合的各种残基。该研究项目将继续调查该酶的调节和结构特征的性质。由于在氰化物中生长的大肠杆菌细胞将在功能上表达植物替代氧化酶，这个细菌系统提供了一种方便的方法来观察引入替代氧化酶蛋白的各种突变的影响。将研究半胱氨酸的定点突变，以确认这些残基的功能。半胱氨酸被认为参与了氧化还原敏感的二硫基/巯基体系和与(-酮酸)的相互作用。此外，随机突变方法加上对被认为作用于喹酚氧化位点的抑制剂的选择，将被用来发现与醌结合有关的残基。为了验证替代氧化酶中是否存在双铁催化位点，将从植物线粒体中提纯该蛋白质，并用于光谱分析。最后，将研究来自真菌的替代氧化酶，以确定是否存在所描述的植物调控特征。虽然植物的“替代氧化酶”存在于线粒体中，而线粒体通常会产生细胞能量，但它所催化的反应实际上浪费了一部分能量。因此，替代氧化酶似乎对植物有害，其活性可能会降低植物的生产力。然而，所有植物都有这种酶的事实表明，替代氧化酶对植物的生存很重要，而且有证据表明，氧化酶在植物对广泛的生物和非生物胁迫的反应中发挥了作用。更好地表征替代酶活性是如何调控的，将有助于理解替代酶在植物新陈代谢中所起的作用，并最终可能导致农业上重要作物的更高植物生产力。

Siedow The cyanide-resistant "alternative" oxidase of plant mitochondria catalyzes the reduction of oxygen to water using electrons derived from the mitochondrial ubiquinone pool. No energy is conserved in this reaction, and thus the function of the alternative oxidase in plant metabolism has been puzzling. Much progress has recently been made in discovering how the activity of the enzyme is regulated in mitochondria. The alternative oxidase has been shown to be dimeric, and its activity is decreased when the two monomers are connected by a disulfide bridge. When this disulfide pair is reduced, the enzyme becomes more active and is able to be stimulated by (-keto acids. The site of action of (-keto acids is likely to be at a sulfhydryl residue, also. Study of the mechanisms of regulation in conjunction with sequence analysis, have provided clues to important structural features of the enzyme. The alternative oxidase is predicted to have a diiron catalytic site, and various residues that may be involved in binding the ubiquinone substrate have been identified. This research project will continue investigation into the nature of the regulatory and structural features of the oxidase. Because Escherichia coli cells grown in cyanide will functionally express the plant alternative oxidase, this bacterial system offers a convenient way to observe the effects of various mutations introduced into the alternative oxidase protein. Site-directed mutations in the cysteines believed to be involved in the redox-sensitive disulfide/sulfhydryl system and interaction with (-keto acids, respectively, will be examined to confirm the function of these residues. Also, a random mutagenesis approach coupled with selection on inhibitors that are presumed to act at the quinol oxidation site will be used to uncover residues involved in quinone binding. To verify the presence of a diiron catalytic site in the alternative oxidase, the protein will be purified from plant mitochondria and used in spectroscopic studies. Final ly, the alternative oxidase from fungi will be studied to determine the presence or absence of the regulatory features described for plants. Although the "alternative oxidase" of plants occurs in the mitochondria where cellular energy is normally produced, the reaction it catalyzes actually wastes some of this energy. Consequently, the alternative oxidase would appear to be harmful to the plant, and its activity might be expected to decrease plant productivity. However, the fact that all plants have this enzyme implies that the alternative oxidase is important to plant survival, and evidence has pointed to a role for the oxidase in plant responses to a wide range of biological and abiological stresses. Better characterization of how alternative oxidase activity is regulated will help in understanding the role the alternative oxidase plays in plant metabolism, and may eventually lead to greater plant productivity for agriculturally important crops.