Studies of the Plant Cyanide-Resistant Alternative Oxidase

植物线粒体的抗氰“替代”氧化酶使用来自线粒体泛醌库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代谢中的功能一直令人困惑。最近在发现线粒体中酶活性如何调节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桥连接时，其活性降低。当这个二硫键对被还原时，酶变得更加活跃，并且能够被α-酮酸刺激。α-酮酸的作用位点也可能在巯基残基上。调控机制的研究与序列分析相结合，为酶的重要结构特征提供了线索。预测替代氧化酶具有二铁催化位点，并且已经鉴定了可能参与结合泛醌底物的各种残基。本研究项目将继续研究氧化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功能性地表达植物替代氧化酶，因此该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。将分别检查据信参与氧化还原敏感性二硫化物/巯基系统以及与β-酮酸相互作用的半胱氨酸中的定点突变，以确认这些残基的功能。此外，一个随机诱变的方法加上对抑制剂的选择，被认为是在醌氧化位点的作用将被用来揭示参与醌结合的残基。为了验证替代氧化酶中存在二铁催化位点，将从植物线粒体中纯化蛋白质并用于光谱研究。最后，将研究来自真菌的替代氧化酶，以确定是否存在针对植物描述的调节特征。 虽然植物的“替代氧化酶”发生在线粒体中，细胞能量通常产生，但它催化的反应实际上浪费了一些能量。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意味着替代氧化酶对植物生存很重要，并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起作用。更好地表征替代氧化酶活性是如何调节的，将有助于理解替代氧化酶在植物代谢中的作用，并可能最终导致农业上重要作物的更高的植物生产力。

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.