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.
