Determination of the stoichiometry of redox-linked proton translocation from the kinetics of pulse experiments. A simulation study.
We have previously published a simple kinetic model to analyse possible pitfalls in kinetic measurements of H+/O ratios in mitochondria [(1984) FEBS Lett. 178, 187-192]. While this model demonstrated how relative electrode response times may affect the results, it did not adequately describe the kinetics of proton back-diffusion across the membrane. Here this model is further developed and improved, and shown to give a good quantitative description of both oxygen-pulse type experiments as well as of experiments where the reaction is started by photolysis of the cytochrome c oxidase-CO complex. Simulations based on this model reveal that the extrapolation procedure used by Lehninger et al. [e.g. (1984) J. Biol. Chem. 259, 4802-4811] to estimate the H+/O ratio will tend to yield overestimated values. This is mainly due to the back-diffusion of protons into the mitochondria, which is not correctly accounted for by this extrapolation.
D002248:Carbon Monoxide / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D004566:Electrodes; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010782:Photolysis; D011522:Protons
D011522:Protons; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV