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, O₂] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of O₂]. In the current study, we used a wide variety of substrates and inhibitors to probe the O₂ sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent K_m for O₂ of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 μm O₂ for the complex I flavin site, complex I electron backflow, complex III Q_O site, and electron transfer flavoprotein quinone oxidoreductase of β-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying O₂]. Based on these data, we hypothesize that at physiological O₂], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high O₂]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental O₂]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.The production of reactive oxygen species (ROS)² by mitochondria has been implicated in numerous disease states, including but not limited to sepsis, solid state tumor survival, and diabetes (1). In addition, mitochondrial ROS (mtROS) play key roles in cell signaling (reviewed in Refs. 2 and 3). There exist within mitochondria several sites for the generation of ROS, with the most widely studied being complexes I and III of the electron transport chain (ETC). However, there is currently some debate regarding the relative contribution of these complexes to overall ROS production (4–9) and the factors that may alter this distribution. One such factor considered herein is O₂]. Estimates of physiological O₂] within tissues (i.e. interstitial O₂]) range from 37 down to 6 μm at 5–40 μm away from a blood vessel (10). More recently, EPR oximetry has estimated tissue O₂] to be in the 12–60 μm range (11). In addition, elegant studies with hepatocytes have shown that O₂ gradients exist within cells, such that an extracellular O₂] of 6–10 μm yields an O₂] of ～5 μm close to the plasma membrane, dropping to 1–2 μm close to mitochondria deep within the cell (12). In cardiomyocytes, at an extracellular O₂] of 29 μm, intracellular O₂] varied in the range 6–25 μm (13). Clearly, different tissues consume O₂ at different rates, so these gradients can vary considerably between tissue and cell types.By definition, the generation of reactive oxygen species by any mechanism, is an O₂-dependent process. However, measurements in intact cells have indicated that mtROS generation increases at lower O₂ levels (1–5% O₂) (14). Proponents of an increase in mtROS in response to hypoxia suggest that under such conditions, reduction of the ETC results in increased leakage of electrons to O₂ at the Q_O site of complex III (14). Such a model posits that increased hypoxic ROS is a mitochondria-autonomous signaling mechanism (i.e. it is an inherent property of the mitochondrial ETC). Therefore, mtROS generation should increase in hypoxia regardless of the experimental system being studied, including isolated mitochondria. In contrast to this hypothesis, we and others have demonstrated that ROS generation by mitochondria is a positive function of O₂] across a wide range of values (0.1–1000 μm O₂) (15–18), suggesting that signaling mechanisms external to mitochondria may be required to facilitate the increased hypoxic mtROS production observed in cells.One limitation of our previous work (15) was that only a single respiratory condition was studied, namely succinate as respiratory substrate (feeding electrons into complex II) plus rotenone to inhibit backflow of electrons through complex I (5, 7). The possibility exists that under different metabolic conditions, which may lead to differential redox states between the cytochromes in the ETC (19, 20), ROS generation may exhibit a different response to O₂]. Thus, in the current study, we examined the response of mtROS generation to O₂] under 11 different conditions, using a variety of respiratory substrates and inhibitors (for a thorough review of electron entry points to the ETC under various substrate/inhibitor combinations, see Ref. 21). Fig. 1 shows a schematic of the mitochondrial ETC, highlighting sites of electron entry resulting from various substrates, binding sites of inhibitors, and major sites of ROS generation. Fig. 2 shows the specific details of each experimental condition, indicating the predicted sites of ROS generation resulting from the use of each substrate/inhibitor combination. The legend to Fig. 2 provides an explanation of each condition.Open in a separate windowFIGURE 1.Mitochondrial pathways of electron flow resulting from the substrates and inhibitors used in this study. Substrates used were glutamate/malate (which generates NADH via the tricarboxylic acid cycle, feeding into complex I), succinate (which feeds electrons directly into complex II), and palmitoyl-carnitine (which feeds electrons into the ETC via acyl-CoA dehydrogenase as well as through the β-oxidation pathway). (For a more thorough explanation, refer to Ref. 21.) Inhibitors used were rotenone (which inhibits at the downstream Q binding site of complex I (9)), malonate (a competitive inhibitor of complex II (25, 26)), and antimycin A (a complex III inhibitor that prevents electron flow to the Q_I site of complex III, thus stabilizing QH^˙ at the Q_O site (6, 28)).Open in a separate windowFIGURE 2.Pathways of electron flow for the substrate/inhibitor combinations used in conditions A–L. Each panel includes the respective maximal respiration rate (VO_{2 max}; nmol of O₂/min/mg of protein) measured under each condition. A, glutamate/malate/malonate. Electrons enter through complex I, whereas electron entry at complex II is inhibited by malonate. ROS generation occurs at the FMN site of complex I as well as the Q_O site of complex III. B, glutamate/malate/malonate/rotenone. Electrons enter through complex I. Electron passage through complex I is inhibited by rotenone binding at the downstream Q site, resulting in maximal ROS production at the FMN site of complex I. ROS production at the Q_O site of complex III is prevented due to no electrons reaching the complex from either complexes I or II, both of which are inhibited. C, glutamate/malate/malonate/antimycin A. Electrons enter through complex I only, since complex II is blocked. Flow of electrons is inhibited by the complex III inhibitor antimycin A, resulting in ROS production at the Q_O site of complex III, as well as the FMN site of complex I. D, succinate. Electrons enter at complex II. ROS is generated by the flow of electrons though the Q_O site of complex III as well as the backflow of electrons through complex I. E, succinate/rotenone. Electrons enter at complex II, and ROS is generated at the Q_O site of complex III, because rotenone is present to inhibit backflow of electrons through complex I. F, succinate/antimycin A. Electrons enter through complex II. ROS is generated at both complex I via backflow and complex III Q_O, with an increased rate at the latter due to inhibition by antimycin A. G, succinate/rotenone/antimycin A. Electrons enter through complex II. Backflow of electrons through complex I is inhibited by rotenone, whereas ROS generation at complex III Q_O is augmented due to the presence of antimycin A. H, glutamate/malate/succinate. Electrons enter at both complexes I and II. ROS is generated from the complex I FMN site and the complex III Q_O site. J, glutamate/malate/succinate/antimycin A. Electrons enter at complexes I and II. ROS generation occurs at the complex I FMN and is augmented at the complex III Q_O site by antimycin A. K, palmitoyl-carnitine. Electrons enter at the ETFQOR. ROS is generated at the ETFQOR as well as complex I via backflow and at the complex III Q_O site. L, palmitoyl-carnitine/rotenone. Electron entry is at the ETFQOR. ROS is generated at the ETFQOR as well as at the complex III Q_O site, whereas ROS due to complex I backflow is blocked by rotenone. Glu, glutamate; Mal, malate; Suc, succinate; PC, palmitoyl-carnitine; Rot, rotenone; AntiA, antimycin A; Malon, malonate.The results of these studies indicated that although ROS generation under all experimental conditions exhibited the same overall response to O₂] (i.e. hyperbolic, with decreased ROS at low O₂]), the apparent K_m for O₂ varied widely between metabolic states.