Multidimensional mitochondrial energetics: Application to the study of electron leak and hydrogen peroxide metabolism

Oxygen consumption is a valuable tool to link with measurements of mitochondrial electron leakage to form reactive oxygen species (ROS), which in mitochondria is predominantly superoxide and H₂O₂. However, oxygen consumption may respond differently to changes in conditions than superoxide/H₂O₂ production does, complicating the use of respiration as a sole indicator of mitochondrial energetics. The same equipment that is valuable for fluorescent monitoring of H₂O₂ efflux provides a straightforward means of estimating membrane potential (ΔΨ), thereby an alternative metric of mitochondrial energetics is readily added to complement studies on the link between mitochondrial energetics and electron leak. By combining multiple aspects of mitochondrial energetics a far more detailed picture emerges on why changes in superoxide/H₂O₂ formation arise with reduced dependence on assumptions. Here we illustrate integration of experimental methods via demonstration of linkages between mitochondrial ΔΨ, oxygen consumption and superoxide/H₂O₂ formation (the latter estimated by H₂O₂ efflux). In doing so we also expand on some pitfalls and cautions for these experimental manipulations of isolated mitochondria and, using these techniques, we raise the possibility that the oxygen affinity for respiration may be higher than the affinity for some sites of electron leak.