Studies of the Cyanide-resistant Alternative Oxidase

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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.