Abstract

Whereas mitochondria are well established as the source of ATP in oxidative phosphorylation (OXPHOS), it is debated if they are also the major cellular sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H₂O₂) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H₂O₂ probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H₂O₂ fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H₂O₂ flux was consistently highest in the Complex II-linked LEAK state, reduced with CI&II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer system capacity. Simultaneous measurement of mitochondrial respiration and H₂O₂ flux requires careful optimization of assay conditions and reveals information on mitochondrial function beyond separate analysis of ROS production. View Full-Text