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.

Estimated O₂ response of ROS generation rate at putative ROS-generating sites within the ETC. Utilizing the data in Fig. 3, the amount of ROS generated for each of the following sites was calculated across a spectrum of [O₂], as detailed under “Experimental Procedures” and calculated according to procedures outlined under “Discussion.” A, the Q_O site of complex III. B, the FMN site of complex I. C, the backflow of electrons through complex I. D, the ETFQOR of β-oxidation. E, the comparison of ROS generation rates using substrates entering at complex I, complex II, or complexes I + II. Inset, ROS generation under these conditions at [O₂] from 0 to 10 μm O₂. For data values obstructed by the inset, see Fig. 3 (conditions A, E, and H). F, the effect of rotenone on the response of mitochondrial ROS production to [O₂] (i.e. data from Fig. 3B minus Fig. 3A). G, the effect of antimycin A on the response of mitochondrial ROS production to [O₂] when respiring on succinate (i.e. data are reproduced from Fig. 3, E (open symbols) and G (filled symbols)).

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)).

O₂ response of ROS generation rate for different substrate inhibitor combinations. ROS generation by isolated rat liver mitochondria under different steady state [O₂] for conditions A–L as detailed in Fig. 2. The substrate/inhibitor combination utilized in each condition is also indicated on each graph. Data are means ± S.E. (n ≥ 5).

ROS generation as a percentage of electron flux through the respiratory chain, as a function of respiration rate (VO₂). Two molecules of O^˙̄₂ are required to make one H₂O₂, so data on the y axis were calculated by dividing 2 × ROS generation rate by the respiration rate. Data are from mitochondria respiring on succinate plus rotenone (condition E) and are taken from the tables in the supplemental material. Error bars are eliminated for clarity. The right-most point is state 4 respiration, with the remaining points on the curve originating from changes in VO₂ due to titration of O₂ levels. The arrows highlight specific values of VO₂ referred to throughout and the extrapolated values of percentage of electrons diverted to ROS.