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.