The physiological role, structure, and regulation of animal alternative oxidases

所有生物都使用电子传输系统(ETS)来产生生长和繁殖所需的能量。真核生物使用线粒体中的ETS来产生ATP，这些ETS的成分在将电子送入链中的络合物和将电子拉出的络合物方面差异很大。除了ETS中典型的末端氧化酶，细胞色素c氧化酶(COX)外，许多生物还含有第二末端氧化酶，称为交替氧化酶(AOX)。虽然AOX也利用氧作为最终的电子载体并产生水，但与COX不同的是，AOX不泵送质子，也不贡献用于产生ATP的质子动力和质子梯度。虽然乍一看，这种反应似乎是在浪费能量，但在许多生物体中，AOX的mRNA和蛋白质水平在环境压力期间会大大增加。这意味着AOX在成功适应应激条件中发挥了作用，可能是通过赋予ETS灵活性。 AOX最初是在植物中发现的，因此大多数研究都是在这个王国进行的。长期以来，人们一直认为动物体内不存在AOX，但过去十年的研究证实，一些动物中存在AOX。我们对AOX在动物体内的分类分布知之甚少，对AOX mRNA在体内的表达及其调控也知之甚少。此外，对AOX蛋白在动物体内的表达和翻译后调控知之甚少。 我们建议使用生物信息学和分子生物学相结合的方法在动物中鉴定新的AOX序列。这将为我们提供更好地理解这种酶的进化史所需的数据。我们还将使用高分辨率呼吸测量法来表征太平洋牡蛎AOX的生化抑制物和激活剂，以更好地了解动物酶的翻译后调节。最后，我们将研究AOX在潮间带桡足类虎虎胁迫耐受性中的生理作用。我们建议使用T.calforNicus作为一种手段，通过实验室实验来研究AOX mRNA和蛋白的表达和调控，这些实验操纵动物所经历的环境条件。我们的工作将是第一次完整的动物AOX的分子和生理特征，并将为从事其他生物工作的研究人员提供一种比较手段。

All organisms use electron transport systems (ETS) to generate energy for growth and reproduction. Eukaryotic organisms use ETS in mitochondria to produce ATP and the components of these ETS are highly variable in terms of the complexes that put electrons into the chain and the complexes that pull electrons out. In addition to the typical terminal oxidase in ETS, cytochrome c oxidase (COX), many organisms contain a second terminal oxidase called alternative oxidase (AOX). While AOX also utilizes oxygen as the final electron carrier and produces water, in contrast to COX, AOX does not pump protons and does not contribute to the proton motive force and proton gradient used to generate ATP. While at first glance this reaction appears to be energetically wasteful, AOX mRNA and protein levels greatly increase during environmental stress in many organisms. This implies that AOX plays a role in the successful acclimation to stress conditions, perhaps by conferring flexibility to the ETS. AOX was initially discovered in plants and therefore most research has been conducted in this kingdom. AOX was long thought to be absent from animals, but work within the past decade has confirmed that it is present in some animals. We have a poor understanding of the taxonomic distribution of AOX in animals and know little about the in vivo expression of AOX mRNA and how it is regulated. Furthermore, little is known about AOX protein expression and post-translational regulation in animals. We propose to identify new AOX sequences in animals using a combination of bioinformatics and molecular biology. This will provide us with the data needed to better understand the evolutionary history of the enzyme. We will also be characterizing the biochemical inhibitors and activators of AOX from Crassostrea gigas (Pacific oyster) by using high-resolution respirometry in order to better understand the post-translational regulation of the animal enzyme. Finally we will be investigating the physiological role of AOX in the stress tolerance of the intertidal copepod Tigriopus californicus. We are proposing to use T. californicus as a means of studying AOX mRNA and protein expression and regulation using laboratory experiments that manipulate the environmental conditions that the animal experiences. Our work will be the first complete molecular and physiological characterization of an animal AOX and will provide a means of comparison for researchers working in other organisms.