The Residual Protonmotive Force in Mitochondria after an Oxygen Pulse

Abstract

Both from irreversible thermodynamics and from mass-action kinetics it can be derived that upon anaerobiosis in an oxygen-pulse experiment the protonmotive force across a mitochondrial membrane undergoes a sudden drop. Under representative conditions the protonmotive force after the drop (the residual protonmotive force) is shown to be less than 3 kJ . [7,11] or 3 [12]. Most of the data are based on a particular type of experiment, i.e. the observation of the number of protons ejected from mitochondria that receive a known amount of substrate (either oxygen, succinate or ATP). A different approach is to compare the phosphorylation potential to the protonmotive force in state-4 mitochondria. This leads to estimation of H+/ATP stoichiometries of 2.5 or higher [13-161. This latter method, however, is not useful for the determination of the H+/O ratios as long as the cytochrome c oxidase reaction cannot be reversed experimentally.An essentially new method for the determination of stoichiometric numbers was developed by taking into account mechanistic parameters when formulating flow-force relations in terms of irreversible thermodynamics [17,18]. Using the derived equations the stoichiometric numbers can in principle be determined under all conditions of oxidation and/or phosphorylation. Initially this method had the disadvantage of being based on the assumption that the relations between reaction/diffusion rates (flows) and their driving forces are proportional. A proof of such proportionality only existed for systems much closer to equilibrium than some processes occurring in mitochondrial oxidative phosphorylation [19]. Investigation of the nature of these flow/force relations for enzyme-catalyzed systems obeying certain, reasonable, boundary conditions [20,21] allowed us to modify the equations in the description, so that it could be applied to mitochondrial oxidative phosphorylation [22].Application of this method enables the experimentor to determine the H+/O and H+/-P stoichiometric numbers under the condition of oxidative phosphorylation, rather than under conditions where the protonmotive force has been eliminated [3 -91. The results of such an application [22] (H+/O = 3.6 for succinate, H + / -P = 2.4) are of course, in agreement with only some of the values determined previously. Evidently, unequivocal results have to emerge from oxygen-pulse experiments before they can be compared with the results obtained with our new quantitative method.In order to try to establish the origin of the conflicting results obtained with oxygen-pulse experiments we set out to reexamine how stoichiometric numbers are derived from oxygen-pulse experiments.In this article we show that, at the time that the oxygen has been consumed, the protonmotive force may drop rapidly and considerably. Thus back extrapolation of the proton re-entrance into the mitochondria is not enough to correct for proton leakage during the oxidation-driven proton extrusion. The values calculated on the basis of such extrapolations must be underestimate...