Under the conditions of low-amplitude mitochondrial swelling, the oxidative phosphorylation system functions in a local coupling mode postulated by Williams in 1961. The proton pumps activation leads to the formation of non-equilibrium membrane bounded proton fraction (nef-H+), which is sorbed on the outer side of the inner mitochondrial membrane under these conditions. This proton fraction is crucial for the ATP synthesis. The present work is devoted to the development of the methods allowing investigations of the properties of nef-H+. For this purpose, a new membranotropic highly hydrophobic uncoupler 2,4,6-trichloro-3-pentadecylphenol was synthesized. In accordance with our results, it can be referred to as a new type of transmemebrane proton carriers, which interact specifically only with nef-H+ outer side of the inner membrane. A new method of the nef-H+ removal from the outer side of the inner mitochondrial membrane under the conditions when the proton pumps are active has been developed. The method is based on neutralization of nef-H+ by hydroxyl anions transferred by H2PO −4 /OH−-antiporter to interfacial border. It was demonstrated that the membrane proton fraction is heterogeneous. Two components of the fraction were identified. And at the same time, it was shown that nucleotide translocator participates in the formation of one component. Also, in this series of experiments it was found that a well-known effect of respiration inhibition by high concentrations of uncoupler under certain conditions can be completely explained by the nef-H+ formation. We presume that one of the most important results of the studies carried out is a new independent evidence of the existence of nef-H+, which is formed under the conditions of proton pump activation.
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Halestrap, A.P., The Regulation of the Matrix Volume of Mammalian Mitochondria in Vivo and in Vitro and Its Role in the Control of Mitochondrial Metabolism, Biochim. Biophys. Acta, 1989, vol. 973, pp. 355–382.
Krasinskaya, I.P., Marshansky, V.N., Dragunova, S.F., and Yaguzhinsky, L.S., Relationships of Respiratory Chain and ATP-Synthetase in Energized Mitochondria, FEBS Lett., 1984, vol. 167, pp. 176–180.
Mitchell, P., Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic Type of Mechanism, Nature, 1961, vol. 191, pp. 144–148.
Williams, R.J., Possible Functions of Chains of Catalysts, J. Theor. Biol., 1961, vol. 1, pp. 1–17.
Antonenko, Y.N., Kovbasnjuk, O.N., and Yaguzhinsky, L.S., Evidence in Favor of the Existence of a Kinetic Barrier for Proton Transfer from a Surface of Bilayer Phospholipid Membrane to Bulk Water, Biochim. Biophys. Acta, 1993, vol. 1150, pp. 45–50.
Yaguzhinsky, L.S., Kostava V.T., Tetenkin V.L., Vitiakova A.A., and Sharyshev A.A., Local Changes of the Acid Concentration in Mitochondrial Membranes, Dokl.AN SSSR (Rus.), 1978, vol. 243, pp. 530–533.
Kovbasnjuk, O.N., Antonenko, Y.N., and Yaguzhinsky, L.S., Proton Dissociation from Nigericin at the Membrane-Water Interface, the Rate-Limiting Step of K+/H+ Exchange on the Bilayer Lipid Membrane, FEBS Lett., 1991, vol. 289, pp. 176–178.
Kozlova, M.V., Gramadskii, K.B., Solodovnikova, I.M., Krasinskaia, I.P., Vinogradov, A.V., and Iaguzhinskii, L.S., Detection and Functional Role of Local Gradients of H+-Ions on the Intracellular Mitochondrial Membrane with Covalently Linked pH-Probe, Biofizika (Rus.), 2003, vol. 48, no. 3, pp. 443–452.
Krasinskaya, I.P., Lapin, M.V., and Yaguzhinsky, L.S., Detection of the Local H+ Gradients on the Internal Mitochondrial Membrane, FEBS Lett., 1998, vol. 440, pp. 223–225.
Solodovnikova, I.M., Iurkov, V.I., Ton’shin, A.A., and Yaguzhinskii, L.S., Local Coupling of Respiration Processes and Phosphorylation in Rat Liver Mitochondria, Biofizika, 2004, vol. 49, no. 1, pp. 47–56.
Yaguzhinsky, L.S., Boguslavsky, L.I., Volkov, A.G., and Rakhmaninova, A.B., Synthesis of ATP Coupled with Action of Membrane Protonic Pumps at the Octane-Water Interface, Nature, 1976, vol. 259, pp. 494–496.
Yaguzhinsky, L.S., Volkov, A.G., and Boguslavsky, L.I., Chlorophenacyl-Inhibitor of the Proton Transfer Step by Membrane Cationic Pumps, Bioelectrochem. Bioenerg., 1977, vol. 4, pp. 225–230.
Yaguzhinsky, L.S., Yurkov, V.I., and Krasinskaya, I.P., On the Localized Coupling of Respiration and Phosphorylation in Mitochondria, Biochim. Biophys. Acta, 2006, vol. 1757, pp. 408–414.
Yurkov, V.I., Fadeeva, M.S., and Yaguzhinsky, L.S., Proton Transfer through the Membrane-Water Interfaces in Uncoupled Mitochondria, Biochemistry (Mosc)., 2005, vol. 70, pp. 195–199.
Drachev, L.A., Kaulen, A.D., and Skulachev, V.P., Correlation of Photochemical Cycle, H+ Release and Uptake, and Electric Events in Bacteriorhodopsin, FEBS Lett., 1984, vol. 178, pp. 331–335.
Heberle, J. and Dencher, N.A., Bacteriorhodopsin in Ice. Accelerated Proton Transfer from the Purple Membrane Surface, FEBS Lett., 1990, vol. 277, pp. 277–280.
Heberle, J., Riesle, J., Thiedemann, G., Oesterhelt, D., and Dencher, N.A., Proton Migration Along the Membrane Surface and Retarded Surface to Bulk Transfer, Nature, 1994, vol. 370, pp. 379–382.
Menger, F.M. and Lynn, J.L., Fast Proton-Transfer at a Micelle Surface, J. Am. Chem. Soc. 1975, vol. 97, pp. 948–949.
Morgan, H., Taylor, D.M., and Oliveira, Jr. O.N., Proton Transport at the Monolayer-Water Interface, Biochim. Biophys. Acta, 1991, vol. 1062, pp. 149–156.
Morgan, H., Taylor, D.M., and Oliveira, Jr. O.N., Two-Dimensional Proton Conduction at a Membrane Surface: Influence of Molecular Packing and Hydrogen Bonding, Chem. Phys. Lett., 1988, vol. 150, pp. 311–314.
Prats, M., Teissie, J., and Tocanne, J.F., Lateral Proton Conduction at Lipid-Water Interfaces and Its Implications for the Chemiosmotic-Coupling Hypothesis, Nature, 1986, vol. 322, pp. 756–758.
Prats, M., Tocanne, J.F., and Teissie, J., Ionization of Phospholipids and Phospholipid-Supported Interfacial Lateral Diffusion of Protons in Membrane Model Systems, Biochim. Biophys. Acta, 1990, vol. 1031, pp. 111–142.
Prats, M., Tocanne, J.F., and Teissie, J., Lateral Proton Conduction at a Lipid/Water Interface. Its Modulation by Physical Parameters. Experimental and Mathematical Approaches, Eur. J. Biochem., 1985, vol. 149, pp. 663–668.
Serowy, S., Saparov, S.M., Antonenko, Y.N., Kozlovsky, W., Hagen, V., and Pohl, P., Structural Proton Diffusion along Lipid Bilayers, Biophys. J., 2003, vol. 84, pp. 1031–1037.
Johnson, D. and Lardy, H., Isolation of Liver or Kidney Mitochondria, Meth. Enzymol., 1967, vol. 10, pp. 94–96.
Krasinskaia, I.P., Litvinov, I.S., Zakharov, S.D., Bakeeva, L.E., and Iaguzhinskii, L.S., Two Qualitatively Different Structuro-Functional States of Mitochondria, Biokhimiia (Rus.), 1989, vol. 54, no. 9, 1550–1556.
Murugova, T.N., Gordeliy, V.I., Kuklin, A.I., Solodovnikova, I.M., and Yaguzhinsky L.S., Study of Three-Dimensionally Ordered Structures of Intact Mitochondria by Small-Angle Neutron Scattering, Crystallography Reports, 2007, vol. 52, no. 3, pp. 545–548.
Andreyev, A., Bondareva, T.O., Dedukhova, V.I., Mokhova, E.N., Skulachev, V.P., and Volkov, N.I., Carboxyatractylate Inhibits the Uncoupling Effect of Free Fatty Acids, FEBS Lett., 1988, vol. 226, pp. 265–269.
Afanas’eva, E.V. and Kostyrko, V.A., Pentachlorophenol Inhibition of Succinate Oxidation by the Respiratory Chain in Submitochondrial Particles from the Bovine Heart, Biokhimiia (Rus.), 1986, vol. 51, no. 5, pp. 823–829.
Kramer R. Structural and Functional Aspects of the Phosphate Carrier from Mitochondria, Kidney Int., 1996, vol. 49, pp. 947–952.
Smirnova, E.G., Kolesova, G.M., Ratnikova, L.A., and Yaguzhinsky, L.S., Hydrophobic Sites of Mitochondrial Electron Transfer System, J. Bioenerget., 1973, vol. 5, pp. 163–174.
Ratnikova, L.A., Yaguzhinskii, L.S., and Skulachev, V.P., Inhibition of Electron Transport in the Respiratory Chain by Phenols with Low Dissociation Constant, Biokhimiia (Rus.), 1971, vol. 36, no. 2, pp. 376–382.
Yaguzhinsky, L.S., Smirnova E.G., Krasinskaya I.P., and Azarenkova T.N., Hydrophobic Sites of the Initial Part of the Mitochondrial Electron-Transporting Chain, Dokl. AN SSSR (Rus.), 1972, vol. 205, pp. 734–738.
Halestrap, A.P., What Is the Mitochondrial Permeability Transition Pore? J. Mol. Cell. Cardiol., 2009, vol. 46, pp. 821–831.
Original Russian Text © K.A. Motovilov, V.I. Yurkov, E.M. Volkov, L.S. Yaguzhinsky, 2009, published in Biologicheskie Membrany, 2009, Vol. 26, No. 5, pp. 408–418.
The article was translated by the authors.
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Motovilov, K.A., Yurkov, V.I., Volkov, E.M. et al. Properties and new methods of non-equilibrium membrane bound proton fraction research under conditions of proton pump activation. Biochem. Moscow Suppl. Ser. A 3, 478 (2009). https://doi.org/10.1134/S1990747809040163
- local coupling
- non-equilibrium states
- new uncoupler
- oxidative phosphorylation