Quantitative analysis of some mechanisms affecting the yield of oxidative phosphorylation:: Dependence upon both fluxes and forces

The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H+ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic propel-ties, regulation, modification of intrinsic stoichiometry). The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation. (1) By manipulating the actual flux through each of the respiratory chain units at constant protonmotive force in yeast mitochondria, we show that the ATP/O ratio decreases when the flux increases. Moreover, the highest efficiency was obtained when the respiratory rate was low and almost entirely controlled by the electron supply. (2) By using almitrine in different kinds of mitochondria, we show that this drug leads to a decrease in ATP synthesis efficiency by increasing the H+/ATP stoichiometry of ATP synthase (Rigoulet M et al. Biochim Biophys Acta 1018. 91-97, 1990). Since this enzyme is reversible, it was possible to test the effect of this drug on the reverse reaction of the enzyme i.e. extrusion of protons catalyzed by ATP hydrolysis, Hence, we are able to prove that, in this case, the decrease in efficiency of oxidative phosphorylation is due to a change in the mechanistic stoichiometry of this proton pump. To our knowledge, this is the first example of a modification in oxidative phosphorylation yield by a change in mechanistic stoichiometry of one of the proton pumps involved. (3) In a model of polyunsaturated fatty acid deficiency in rat, it was found that non-ohmic proton leak was increased, while ohmic leak was unchanged. Moreover, an increase in redox slipping was also involved, leading to a complex picture. However, the respective role of these two mechanisms may be deduced from their intrinsic properties. For each steady state condition, the quantitative effect of these two mechanisms in the decrease of oxidative phosphorylation efficiency depends on the values of different fluxes or forces involved. (4) Finally the comparison of the thermokinetic data in view of the three dimensional-structure of some pumps (X-ray diffraction) also gives some information concerning the putative mechanism of coupling (i.e. redox loop or proton pump) and their kinetic control versus regulation of mitochondrial oxidative phosphorylation.