ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles

Abstract

The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3′- O-(3-( N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD + were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) — an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276–287) — when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD + required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the Δ \\ ̃ gm H + . In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al.