Molecular mechanistic insights into uncoupling of ion transport from ATP synthesis

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1Fo-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.