This paper is an overview of the theoretical and experimental studies performed in our laboratory to answer the question whether there exist conditions where the hypothetical mechanism of the localized coupling of respiration and phosphorylation postulated by R. Williams in 1961 operates. These studies were undertaken to verify the earlier suggestion that mitochondria may exist in two structural and functional states. Correspondingly, there are two operation modes of oxidative phosphorylation, one of which corresponds to the Williams' mechanism of localized coupling and the other, to the Mitchell's mechanism of delocalized coupling. The paper considers the principle of the energy conservation of oxidative reactions in mitochondrial membranes in the form of the thermodynamic potential of hydrogen ions (Deltamusol) lacking, in part, the solvation shell. We present experimental evidence for the existence of the mechanism of localized coupling and describes the conditions favorable for its implementation. The experiments described in this paper show that the aforementioned models for proton coupling are not necessarily alternative. A conclusion is made that, depending on the particular conditions, either localized or delocalized coupling mechanisms of oxidative phosphorylation may come into operation.
Increase in maximal respiration rate of uncoupled mitochondria in response to increase in concentration of non-penetrating buffer has been demonstrated. This phenomenon did not depend on chemical structure of uncouplers and composition of the non-penetrating buffer. Use of covalently attached pH probe, FITC, revealed that at low buffer concentration (3 mM) the H(+)-pump functioning results in local increase in proton concentration on the outer surface of the inner mitochondrial membranes. In other words, local H(+) gradient was generated. Increase in buffer concentration up to 20 mM caused sharp decrease in this gradient, which occurred in parallel to increase in the respiration rate. It is concluded that both effects described here may be attributed to the process of proton transfer through the interfaces of the mitochondrial membrane: the rate of respiratory H(+) pumps of uncoupled mitochondria under conditions of low buffer capacity of medium is limited by the stage of proton release from outer surface of the coupling membrane. The inhibition mechanism of respiration by high concentrations of uncouplers is also discussed.
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