Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions

The mitochondrial generation of reactive oxygen species (ROS) plays a central role in many cell signaling pathways, but debate still surrounds its regulation by factors, such as substrate availability, [O-2] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of [O-2]. In the current study, we used a wide variety of substrates and inhibitors to probe the O-2 sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent K-m for O-2 of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 mu M O-2 for the complex I flavin site, complex I electron backflow, complex IIIQ(O) site, and electron transfer flavoprotein quinone oxidoreductase of beta-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying [O-2]. Based on these data, we hypothesize that at physiological [O-2], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high [O-2]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental [O-2]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.