RESUMEN
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 pHâ¯5 to pHâ¯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.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Catálisis , Diarilquinolinas/química , Diarilquinolinas/metabolismo , Diarilquinolinas/farmacología , Dinitrofenoles/química , Dinitrofenoles/metabolismo , Concentración de Iones de Hidrógeno , Transporte Iónico , Cinética , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/química , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Fosforilación OxidativaRESUMEN
A wealth of molecular mechanistic insights has been provided into the coupling of ion transport to ATP synthesis based on a two-ion theory of biological energy coupling. A kinetic scheme that considers the mode of functioning of a single F1FO-ATP synthase molecule with H+-A- cotransport and unidirectional rotation of the c-rotor in the membrane-bound FO-portion of the enzyme has been developed. Mathematical analysis leads to a detailed enzyme kinetic model applicable to a population of molecules which is compared with experimental data on the pH dependence of ATP synthesis. The model agrees well with the experimental data, and a single equation with a single set of standard enzymological kinetic parameters has been shown to explain the experimental data over the entire range of conditions for the chloroplast ATP synthase. The analysis gives novel insights into kinetic and mechanistic characteristics of ATP synthesis in FO. These include an order imposed on ion binding and unbinding events in FO, the essential role of the anion in direct activation of the ATP synthase (in addition to its role as a permeant ion), and the integration in a novel way of the functions of cooperativity and cotransport of dicarboxylic acid anions and protons during physiological ATP synthesis. Further, Wyman's pioneering classical work on the thermodynamics of linked functions has been shown to offer a new approach to distinguish between various models of energy coupling in ATP synthesis. All these results have been found to be inconsistent with Mitchell's chemiosmotic theory and are shown to be in agreement with Nath's torsional mechanism of energy transduction and ATP synthesis.