A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation

The alternative oxidase is a quinol oxidase of the respiratory chain of pla nts and some fungi and protists. Its activity is regulated by redox-sensiti ve disulphide bond formation between neighbouring subunits and direct inter action with certain alpha-ketoacids. To investigate these regulatory mechan isms, we undertook site-directed mutagenesis of soybean and Arabidopsis alt ernative oxidase cDNAs, and expressed them in tobacco plants and Escherichi a coli, respectively, The homologous C99 and C127 residues of GmAOX3 and At AOX1a, respectively, were changed to serine, Tn the plant system, this subs titution prevented oxidative inactivation of alternative oxidase and render ed the protein insensitive to pyruvate activation, in agreement with the re cent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 2 73, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. Howe ver, the mutated protein is instead activated specifically by succinate, Me asurements of AtAOX1a activity in bacterial membranes lacking succinate deh ydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidas e proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implicatio ns for the efficiency of respiration in the tissues which express them. (C) 1999 Federation of European Biochemical Societies.