Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions
2009; Elsevier BV; Volume: 284; Issue: 24 Linguagem: Inglês
10.1074/jbc.m809512200
ISSN1083-351X
AutoresDavid L. Hoffman, Paul S. Brookes,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoThe 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 Km 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 QO 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 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 Km 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 QO 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) 2The abbreviations used are: ROSreactive oxygen speciesmtROSmitochondrial ROSETCelectron transport chainETFQORelectron transfer flavoprotein quinone oxidoreductase. by mitochondria has been implicated in numerous disease states, including but not limited to sepsis, solid state tumor survival, and diabetes (1Bayir H. Kagan V.E. Crit. Care. 2008; 12: 206Crossref PubMed Scopus (121) Google Scholar). In addition, mitochondrial ROS (mtROS) play key roles in cell signaling (reviewed in Refs. 2Acker H. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2005; 360: 2201-2210Crossref PubMed Scopus (54) Google Scholar and 3Brookes P.S. Yoon Y. Robotham J.L. Anders M.W. Sheu S.S. Am. J. Physiol. Cell Physiol. 2004; 287: C817-C833Crossref PubMed Scopus (1954) Google Scholar). 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 (4Chen Q. Vazquez E.J. Moghaddas S. Hoppel C.L. Lesnefsky E.J. J. Biol. Chem. 2003; 278: 36027-36031Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar, 5St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. Chem. 2002; 277: 44784-44790Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 6Muller F.L. Roberts A.G. Bowman M.K. Kramer D.M. Biochemistry. 2003; 42: 6493-6499Crossref PubMed Scopus (118) Google Scholar, 7Muller F.L. Liu Y. Abdul-Ghani M.A. Lustgarten M.S. Bhattacharya A. Jang Y.C. Van Remmen H. Biochem. J. 2008; 409: 491-499Crossref PubMed Scopus (128) Google Scholar, 8Adam-Vizi V. Chinopoulos C. Trends Pharmacol. Sci. 2006; 27: 639-645Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 9Lambert A.J. Brand M.D. J. Biol. Chem. 2004; 279: 39414-39420Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) 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 (10Tsai A.G. Johnson P.C. Intaglietta M. Physiol. Rev. 2003; 83: 933-963Crossref PubMed Scopus (361) Google Scholar). More recently, EPR oximetry has estimated tissue [O2] to be in the 12–60 μm range (11Matsumoto A. Matsumoto S. Sowers A.L. Koscielniak J.W. Trigg N.J. Kuppusamy P. Mitchell J.B. Subramanian S. Krishna M.C. Matsumoto K. Magn. Reson. Med. 2005; 54: 1530-1535Crossref PubMed Scopus (74) Google Scholar). 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 (12Jones D.P. Am. J. Physiol. Cell Physiol. 1986; 250: C663-C675Crossref PubMed Google Scholar). In cardiomyocytes, at an extracellular [O2] of 29 μm, intracellular [O2] varied in the range 6–25 μm (13Takahashi E. Sato K. Endoh H. Xu Z.L. Doi K. Am. J. Physiol. Heart Circ. Physiol. 1998; 275: H225-H233Crossref PubMed Google Scholar). Clearly, different tissues consume O2 at different rates, so these gradients can vary considerably between tissue and cell types. reactive oxygen species mitochondrial ROS electron transport chain electron transfer flavoprotein quinone oxidoreductase. 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) (14Guzy R.D. Schumacker P.T. Exp. Physiol. 2006; 91: 807-819Crossref PubMed Scopus (685) Google Scholar). 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 QO site of complex III (14Guzy R.D. Schumacker P.T. Exp. Physiol. 2006; 91: 807-819Crossref PubMed Scopus (685) Google Scholar). 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) (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar, 16Boveris A. Chance B. Biochem. J. 1973; 134: 707-716Crossref PubMed Scopus (2112) Google Scholar, 17Mairbaurl H. Hotz L. Chaudhuri N. Bartsch P. MiP 2005: Abstracts of the 4th Mitochondrial Physiology Meeting, Schroeken, Austria, September 16–20, 2005. MiPNet Publications, Innsbruck, Austria2005: 26-27Google Scholar, 18Michelakis E.D. Hampl V. Nsair A. Wu X. Harry G. Haromy A. Gurtu R. Archer S.L. Circ. Res. 2002; 90: 1307-1315Crossref PubMed Scopus (257) Google Scholar), 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 (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar) 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 (5St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. Chem. 2002; 277: 44784-44790Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 7Muller F.L. Liu Y. Abdul-Ghani M.A. Lustgarten M.S. Bhattacharya A. Jang Y.C. Van Remmen H. Biochem. J. 2008; 409: 491-499Crossref PubMed Scopus (128) Google Scholar). The possibility exists that under different metabolic conditions, which may lead to differential redox states between the cytochromes in the ETC (19Campian J.L. Qian M. Gao X. Eaton J.W. J. Biol. Chem. 2004; 279: 46580-46587Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 20Aw T.Y. Wilson E. Hagen T.M. Jones D.P. Am. J. Physiol. Renal Physiol. 1987; 253: F440-F447Crossref PubMed Google Scholar), 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. 21Gnaiger E. Mitochondrial Pathways and Respiratory Control. 2nd Ed. MiPNet Publications, Innsbruck, Austria2007Google Scholar). 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.FIGURE 2Pathways of electron flow for the substrate/inhibitor combinations used in conditions A–L. Each panel includes the respective maximal respiration rate (VO2 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 QO 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 QO 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 QO 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 QO 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 QO 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 QO, 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 QO 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 QO 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 QO 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 QO site. L, palmitoyl-carnitine/rotenone. Electron entry is at the ETFQOR. ROS is generated at the ETFQOR as well as at the complex III QO 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 Km for O2 varied widely between metabolic states. All chemicals were the highest grade available from Sigma unless otherwise indicated. Male adult Sprague-Dawley rats (250 g) were purchased from Harlan (Indianapolis, IN) and were maintained in accordance with Ref. 53National Research CouncilGuide for Care and Use of Laboratory Animals. National Institutes of Health Publication 86-23, National Institutes of Health, Bethesda, MD1996Google Scholar. All procedures were approved by the University of Rochester Committee on Animal Resources (protocol number 2007-087). Liver mitochondria were isolated by differential centrifugation, as described previously (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar). Mitochondrial incubations were performed using an open flow respirometry cell, as described previously (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar, 22Brookes P.S. Kraus D.W. Shiva S. Doeller J.E. Barone M.C. Patel R.P. Lancaster Jr., J.R. Darley-Usmar V. J. Biol. Chem. 2003; 278: 31603-31609Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Briefly, mitochondria were suspended in the liquid phase in a stirred chamber with a head space gas of tightly controlled pO2 flowing above. Such a system, in which the liquid phase [O2] is measured with an O2 electrode, permits prolonged mitochondrial incubation at tightly controlled steady-state [O2] and the calculation of mitochondrial O2 consumption by a simplified Fick equation (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar, 22Brookes P.S. Kraus D.W. Shiva S. Doeller J.E. Barone M.C. Patel R.P. Lancaster Jr., J.R. Darley-Usmar V. J. Biol. Chem. 2003; 278: 31603-31609Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 23Cole R.P. Sukanek P.C. Wittenberg J.B. Wittenberg B.A. J. Appl. Physiol. 1982; 53: 1116-1124Crossref PubMed Scopus (39) Google Scholar). The O2 electrode was calibrated daily with air-saturated deionized H2O, with or without sodium dithionite. The impact of additions to mitochondrial incubations (e.g. substrates or inhibitors) on O2 solubility was no more than 0.4% of the total. A fiber optic fluorimeter was built into the chamber, permitting measurement of mitochondrial ROS generation using the H2O2-sensitive dye Amplex red (23Cole R.P. Sukanek P.C. Wittenberg J.B. Wittenberg B.A. J. Appl. Physiol. 1982; 53: 1116-1124Crossref PubMed Scopus (39) Google Scholar). Authentic H2O2 was added at the end of each experimental run to internally calibrate the fluorescent signal. Such a method ensures that the obtained signal truly reflects the net H2O2 production and is not affected by scavenging due to enzymes, such as catalase. Incubations were carried out in mitochondrial respiration buffer (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar), with oligomycin (1 μg/ml) present to enforce state 4 respiration. Where indicated, mitochondrial substrates and inhibitors were used at the following concentrations: glutamate (10 mm), malate (5 mm), succinate (10 mm), palmitoyl-carnitine (1 μm), rotenone (1 μm), antimycin A (10 μm), malonate (2 mm). They were present from the beginning of incubations before mitochondrial addition. Superoxide dismutase (80 units/ml) was present in all incubations to ensure rapid dismutation of O⨪2 to H2O2 and to avoid scavenging of the former by reaction with nitric oxide (NO·). This was a precaution, despite our previous observations that additional superoxide dismutase was not necessary in this regard and that NO· scavenging of O⨪2 (which would lead to peroxynitrite-mediated tyrosine nitration) was not occurring in hypoxia (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar). The latter is also unlikely because the Km for O2 of all NOS isoforms is very high (6–24 μm) (24Rengasamy A. Johns R.A. J. Pharmacol. Exp. Ther. 1996; 276: 30-33PubMed Google Scholar), so NO· generation actually decreases in hypoxia. Full details on each combination of substrates/inhibitors and the putative sites of ROS generation resulting from each are given under "Results" and in the legends to FIGURE 1, FIGURE 2. The steady-state [O2] reached in open flow respirometry is not an independent variable but a result of the individual characteristics of each mitochondrial incubation. Therefore, it is not possible to use the raw data to calculate average rates of ROS generation at a single [O2]. Thus, for each metabolic condition, the empirical values of ROS generation across a range of steady-state [O2] (typically 7–10 points/curve) were fitted to a single-substrate binding curve, employing Prism software (GraphPad, San Diego, CA), as described previously (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar). The curve fit parameters (Vmax, Km) were then used to extrapolate ROS generation rates at common values of [O2], and these data then averaged between individual experiments (n ≥ 5). State 4 respiration rates (VO2; nmol of O2/min/mg of protein) under each metabolic condition were calculated across the range of [O2] values studied, as previously described (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar, 22Brookes P.S. Kraus D.W. Shiva S. Doeller J.E. Barone M.C. Patel R.P. Lancaster Jr., J.R. Darley-Usmar V. J. Biol. Chem. 2003; 278: 31603-31609Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The maximal VO2 (at high [O2]) for each condition is listed in each panel of Fig. 2, whereas the full response curves of VO2 to [O2] are in Table S1. VO2 varied considerably between metabolic substrates. For example, a higher VO2 was observed with complex II substrates (condition E) than with complex I substrates (condition A). The consensus view is that because fewer H+ are pumped across the inner membrane when electrons enter at complex II, the ETC has to work faster in condition E (and thus consume more O2) to maintain the same H+ gradient as in condition A. The generation of ROS as a function of [O2] for metabolic conditions A–L (Fig. 2) is illustrated in Fig. 3. Respiring on complex I-linked substrates (glutamate plus malate in the presence of malonate to inhibit complex II), ROS generation was maximal at 250 pmol/min/mg mitochondrial protein, whereas Km was 0.25 μm O2 (Fig. 3A). As expected, the addition of rotenone, which inhibits at the downstream Q site of complex I, increased maximal ROS slightly (Vmax 295) while causing a right shift in the curve (Km = 2.0 μm O2; Fig. 3B). Similarly, inhibition at complex III by antimycin A also increased ROS (Vmax 460) and further right-shifted the curve (Km = 5.0 μm O2; Fig. 3C).FIGURE 3O2 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) With succinate as the respiratory substrate, feeding electrons into complex II (Fig. 3D), maximal ROS generation was 330 pmol/min/mg mitochondrial protein with a Km of 1.8 μm O2. Some of this ROS was due to backflow of electrons through complex I, since the addition of rotenone (Fig. 3E) brought the Vmax value down to 105 and the Km to 0.7 μm O2. Similarly to the situation with complex I-linked substrates (see above), the addition of antimycin A to succinate-respiring mitochondria (Fig. 3F) raised maximal ROS generation to 420 pmol/min/mg mitochondrial protein and strongly right-shifted the curve (Km = 12 μm O2). Adding both rotenone and antimycin A together (Fig. 3G) gave a Vmax of 380 and a Km of 4 μm O2. Thus, in both complex I- and II-linked respiration, antimycin A-induced ROS generation is heavily O2-dependent, having a much greater Km than base-line ROS generation (Fig. 3, C versus A, G versus E, and F versus D). Under conditions of dual electron entry at complexes I and II (i.e. respiration on glutamate, malate, and succinate together) (Fig. 3H), maximal ROS generation was 330 pmol/min/mg mitochondrial protein, and Km was 0.5 μm O2. As seen for complex I- or complex II-linked substrates alone, the addition of antimycin A to the dual electron entry condition (Fig. 3J) resulted in the highest ROS generation measured under any condition (Vmax = 490) and a strongly right-shifted curve (Km = 9 μm O2). In mitochondria respiring on palmitoyl-carnitine, maximal ROS was 290 pmol/min/mg mitochondrial protein, with a Km of 1.0 μm O2. Similar to the situation with complex II, some of this ROS may result from complex I backflow, since the addition of rotenone resulted in a decrease in ROS (Vmax = 250) and a right shift in the curve (Km = 4 μm O2). 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 (Fig. 2, A–L). Fig. 3 shows mtROS generation as a function of [O2] for each of the 11 conditions A–L. In conditions A–C, electrons entered the ETC at complex I, with complex II blocked by malonate (25Wojtovich A.P. Brookes P.S. Biochim. Biophys. Acta. 2008; 1777: 882-889Crossref PubMed Scopus (92) Google Scholar, 26Pardee A.B. Potter V.R. J. Biol. Chem. 1949; 178: 241-250Abstract Full Text PDF PubMed Google Scholar). In conditions D–G, electrons entered at complex II. In conditions H and J, electrons entered at both complexes I and II, and in conditions K and L, electrons entered at the β-oxidation electron transfer flavoprotein quinone oxidoreductase (ETFQOR). Despite the different sites of electron entry, all conditions exhibited the same overall pattern of ROS generation in response to [O2], namely a hyperbolic function with lower ROS generation rate at lower [O2]. Thus, it appears that our previous data set showing decreased mtROS at low [O2] (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar) was not an artifact of the metabolic conditions chosen (succinate plus rotenone). Although information on ROS generation under different substrate/inhibitor conditions is useful in the field of isolated mitochondrial bioenergetics, it would be more useful to know the O2 sensitivity of ROS generation from putative sites within the ETC. Thus, a series of calculations was devised to estimate ROS generation from each of four putative sites, at varying [O2] (Fig. 4). Below, the rationale behind each calculation is discussed along with the results. The rate of ROS generation from the QO site of complex III was estimated by two methods. First, it was estimated by using the rate of ROS generation obtained in the presence of succinate as substrate (complex II) plus rotenone to inhibit electron backflow through complex I (i.e. condition E) (5St-Pierre J. Buckingham J.A. Roebuck S.J. Brand M.D. J. Biol. Chem. 2002; 277: 44784-44790Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 9Lambert A.J. Brand M.D. J. Biol. Chem. 2004; 279: 39414-39420Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 27Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1367) Google Scholar). Under this condition, ROS generation occurs primarily at the complex III QO site (6Muller F.L. Roberts A.G. Bowman M.K. Kramer D.M. Biochemistry. 2003; 42: 6493-6499Crossref PubMed Scopus (118) Google Scholar, 7Muller F.L. Liu Y. Abdul-Ghani M.A. Lustgarten M.S. Bhattacharya A. Jang Y.C. Van Remmen H. Biochem. J. 2008; 409: 491-499Crossref PubMed Scopus (128) Google Scholar, 28Sun J. Trumpower B.L. Arch. Biochem. Biophys. 2003; 419: 198-206Crossref PubMed Scopus (136) Google Scholar). Second, the rate of ROS generation due to backflow of electrons through complex I (calculated below) was subtracted from the rate of ROS with succinate alone in which electrons flow both forward through complex III and backward through complex I (condition D). The two values for complex III QO site ROS generation were then averaged (Fig. 4A), resulting in a Vmax of 150 and apparent Km of 2.0. To estimate ROS generation by the complex I FMN site, we used mitochondria respiring on complex I-linked substrates alone (i.e. glutamate plus malate) in the presence of a complex II inhibitor to prevent electron entry due to passage of substrates through the tricarboxylic acid cycle. The inhibitor chosen was malonate, since 2-thenoyltrifluoroacetone (29Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar) exhibited an absorbance spectrum that interfered with Amplex Red (not shown) and may also stimulate ROS generation at complex II (30Byun H.O. Kim H.Y. Lim J.J. Seo Y.H. Yoon G. J. Cell. Biochem. 2008; 104: 1747-1759Crossref PubMed Scopus (56) Google Scholar, 31Chen Y. McMillan-Ward E. Kong J. Israels S.J. Gibson S.B. J. Cell Sci. 2007; 120: 4155-4166Crossref PubMed Scopus (364) Google Scholar). Under condition A (glutamate, malate, and malonate), some electron flux proceeds via the Q pool to complex III. Thus, it is necessary to subtract ROS generation by the complex III QO site. Furthermore, it is insufficient to merely subtract ROS as calculated above, since that flux was calculated from mitochondria respiring on succinate, and the flux through the respiratory chain (i.e. VO2) was lower with glutamate plus malate. This lower electron flux has two opposing effects on ROS generation by complex III QO. First, fewer electrons reach complex III, as shown by the VO2 in condition A (Fig. 2A), which was 39.3% of that in condition E (Fig. 2E). Second, this slower electron flux through complex III results in an increased dwell time for the ubisemiquinone radical at the QO site, which enhances ROS generation (32Forquer I. Covian R. Bowman M.K. Trumpower B.L. Kramer D.M. J. Biol. Chem. 2006; 281: 38459-38465Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To correct for this second effect, it is necessary to determine the relationship between the percentage of electrons diverted to ROS and the total electron flux (VO2). Fig. 5 shows this relationship for mitochondria respiring in condition E (succinate plus rotenone), indicating that a VO2 of 19.5 nmol of O2/min/mg results in 1.065% of electrons going to ROS. Lowering the VO2 to 7.7 (i.e. the VO2 in condition A) increases this value to 1.817%. Thus, the percentage of electrons diverted to ROS is 1.71-fold greater in condition A versus condition E. Combining these two correction factors (39.3% × 1.71) indicates that it is necessary to subtract 67.2% of the ROS from the complex III QO site (Fig. 4A) to reveal the residual ROS from the complex I FMN site. The result is shown in Fig. 4B, and interestingly, the shape of the curve is not a classical hyperbolic function but instead indicates a Vmax of 170 at 5 μm O2, with ROS declining very slightly at higher [O2]. The apparent Km from this curve was estimated as 0.19 μm O2. These data therefore suggest that the complex I FMN site is able to generate ROS at O2 levels far below that at which the complex III QO site is already O2-limited (Km = 2.0 μm O2; see Fig. 4A). Notably, Fig. 5, which shows that the percentage of electron flux diverted to ROS increases as respiration slows down, might be misconstrued as demonstrating that mitochondrial ROS generation increases at low respiration rates (such as those caused by low [O2]). However, as we previously discussed (15Hoffman D.L. Salter J.D. Brookes P.S. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H101-H108Crossref PubMed Scopus (135) Google Scholar), although a greater percentage of electrons m
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