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

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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.