Feedback Interactions in the Control of Citric Acid Cycle Activity in Rat Heart Mitochondria

Factors regulating the citric acid cycle have been investigated in rat heart mitochondria oxidizing pyruvate plus malate or acetylcarnitine plus malate as substrates. Effects caused by changing the NAD oxidation-reduction state, the intramitochondrial ATP:ADP ratio, and the respiratory rate were studied in five different metabolic states produced by additions of ADP, oligomycin, or uncouplers. The accumulations of cycle intermediates and the mitochondrial content of CoA derivatives and pyridine nucleotides were measured in extracts prepared from the whole incubation medium or after rapid separation of the mitochondria from the medium. These data, together with measurements of substrate utilization, were used to calculate flux through the individual steps of the citric acid cycle. Pyruvate dehydrogenase was found not to be rate limiting for citric acid cycle activity. Flux through citrate synthase in State 3 was approximately the same (100 nmoles per min per mg) whether pyruvate or acetylcarnitine were used to generate acetyl-CoA, whereas acetyl-CoA levels were higher with pyruvate as substrate. Flux in State 4 with either substrate was diminished by 75 to 85% relative to State 3 and was associated during early incubation times with elevated levels of both NADH and acetyl-CoA, consonant with regulation of pyruvate dehydrogenase by product inhibition. A comparison of flux through citrate synthase and changes in the levels of intramitochondrial malate, citrate, ATP, and the NAD oxidation-reduction state showed that increased flux associated with a State 4 to 3 transition could be accounted for largely by an increased availability of oxalacetate to citrate synthase rather than by ATP inhibition. On the other hand, comparisons between states in which phosphate acceptor was not rate limiting for electron transport (State 3, uncoupled or uncoupled plus oligomycin) showed that oxalacetate availability remained high because of the highly oxidized state of the pyridine nucleotides, and indicated a regulation of citrate synthase under conditions of low acetyl-CoA availability by an energy-linked process dependent on substrate level phosphorylation. Thus, a 55% inhibition of flux through citrate synthase was obtained after addition of oligomycin to the uncoupled state with acetylcarnitine plus malate as substrate, but only a 25% inhibition was achieved with pyruvate plus malate as substrate. The intramitochondrial ATP:ADP ratio increased from 0.3 to 4:6 with either substrate under these conditions. A kinetic evaluation of the data indicated that the energy-dependent inhibition was not caused by a direct effect of ATP on citrate synthase, but was due to changes of the succinyl-CoA content relative to that of acetyl-CoA. This conclusion is based (a) on the finding of an accumulation of a short chain acyl-CoA compound identifiable as succinyl-CoA by direct enzyme assay under conditions of diminished citrate synthase flux, (b) a correlation between decreased flux and lowered acetyl-CoA levels in the different experiments, and (c) the observation that succinyl-CoA is an inhibitor of citrate synthase competitive with acetyl-CoA. A comparison of succinyl-CoA levels in mitochondria incubated in the different metabolic states indicated that its content was regulated by the phosphorylation state of the adenine nucleotides, presumably via the effect of altered GTP:GDP ratios on succinate thiokinase. Product inhibition of α-ketoglutarate dehydrogenase by succinyl-CoA was evidenced from an inverse relationship between α-ketoglutarate accumulation and succinyl-CoA levels. The conclusion is reached that feedback from the electron transport chain to the citric acid cycle is mediated by a combination of factors which include the phosphorylation state of the adenine and guanine nucleotides and the oxidation-reduction state of the pyridine nucleotides. Changes of these parameters secondarily affect the intramitochondrial concentrations of oxalacetate, acetyl-CoA, and succinyl-CoA, which are the direct regulators of citrate synthase activity.