ALTERNATIVE OXIDASE ACTIVITY IN TOBACCO LEAF MITOCHONDRIA - DEPENDENCE ON TRICARBOXYLIC-ACID CYCLE-MEDIATED REDOX REGULATION AND PYRUVATE ACTIVATION

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high lev els of mitochondrial alternative oxidase (AOX) protein due to the intr oduction of a sense transgene(s) of Aox1, the nuclear gene encoding AO X, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX prote in was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and m ore active dimer) and its subsequent activation by certain alpha-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may b e specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgen ic leaves lacking AOX due to the introduction of an Aox1 antisense tra nsgene or multiple sense transgenes were used to investigate the poten tial physiological significance of the AOX-regulatory mechanisms. Unde r conditions in which respiratory carbon metabolism is restricted by t he capacity of mitochondrial electron transport, feed-forward activati on of AOX by mitochondrial reducing power and pyruvate may act to prev ent redirection of carbon metabolism, such as to fermentative pathways .