The oxidation of citrate, isocitrate and cis-aconitate by isolated mitochondria.

There are five oxidative steps involved in the conversion of pyruvate into carbon dioxide and water in the tricarboxylic acid cycle; four of these steps lead to the reduction of nicotinamide nucleotide coenzyme more or less directly. The step catalysed by the succinate dehydrogenase results in the 'energy-linked' reduction of nicotinamide nucleotide coenzyme, under certain conditions (Chance & Hollunger, 1960), but this process is obviously very different from those reductions that occur in the other oxidative events. Two of these latter reactions, involving pyruvate dehydrogenase and oc-oxoglutarate dehydrogenase, require amongst other cofactors lipoate or a derivative (see Krebs & Kornberg, 1957). It appears that the reduced lipoate is re-oxidized by NAD and that this reaction is catalysed by the lipoate dehydrogenase (Hager & Gunsalus, 1953; Cutolo, 1956). The two remaining oxidative steps, utilizing isocitrate dehydrogenase and malate dehydrogenase, are thought to lead to the reduction of nicotinamide nucleotide coenzyme directly. The experimental results given in the present paper lead to the conclusion that the nicotinamide nucleotide coenzyme reduced by the isocitrate dehydrogenase is not available to the respiratory chain and that, despite the presence ofNAD and an active nicotinamide nucleotide transhydrogenase, the reduced nucleotide must react with oxaloacetate, a process catalysed by malate dehydrogenase. The malate is then oxidized by reaction with nicotinamide nucleotide coenzyme, which is, in this case, available to the cytochrome system (Chappell, 1961). The rate at which citrate is oxidized appears to be limited by mitochondrial aconitase activity. This is not the case when C68aconitate serves as substrate. The significance of these findings is discussed in relation to the structural organization of the mitochondrion.