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

Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 24, pp. 16236 –16245, June 12, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species □ S Generation Depends on Metabolic Conditions Received for publication, December 18, 2008, and in revised form, March 18, 2009 Published, JBC Papers in Press, April 14, 2009, DOI 10.1074/jbc.M809512200 ‡ §1 David L. Hoffman and Paul S. Brookes ‡ § From the Departments of Biochemistry and Anesthesiology, University of Rochester Medical Center, Rochester, New York 14642 The mitochondrial generation of reactive oxygen species blood vessel (10). More recently, EPR oximetry has estimated (ROS) plays a central role in many cell signaling pathways, but tissue [O ] to be in the 12–60 M range (11). In addition, ele- debate still surrounds its regulation by factors, such as substrate gant studies with hepatocytes have shown that O gradients availability, [O ] and metabolic state. Previously, we showed exist within cells, such that an extracellular [O ]of6–10 M that in isolated mitochondria respiring on succinate, ROS gen- yields an [O ]of 5 M close to the plasma membrane, drop- eration was a hyperbolic function of [O ]. In the current study, ping to 1–2 M close to mitochondria deep within the cell (12). we used a wide variety of substrates and inhibitors to probe the In cardiomyocytes, at an extracellular [O ]of29 M, intracel- O sensitivity of mitochondrial ROS generation under different lular [O ] varied in the range 6–25 M (13). Clearly, different metabolic conditions. From such data, the apparent K for O of tissues consume O at different rates, so these gradients can m 2 putative ROS-generating sites within mitochondria was esti- vary considerably between tissue and cell types. mated as follows: 0.2, 0.9, 2.0, and 5.0 M O for the complex I By definition, the generation of reactive oxygen species by any flavin site, complex I electron backflow, complex III Q site, and mechanism, is an O -dependent process. However, measure- electron transfer flavoprotein quinone oxidoreductase of -ox- ments in intact cells have indicated that mtROS generation idation, respectively. Differential effects of respiratory inhibi- increases at lower O levels (1–5% O ) (14). Proponents of an 2 2 tors on ROS generation were also observed at varying [O ]. increase in mtROS in response to hypoxia suggest that under Based on these data, we hypothesize that at physiological [O ], such conditions, reduction of the ETC results in increased leak- complex I is a significant source of ROS, whereas the electron age of electrons to O at the Q site of complex III (14). Such a 2 O transfer flavoprotein quinone oxidoreductase may only con- model posits that increased hypoxic ROS is a mitochondria- tribute to ROS generation at very high [O ]. Furthermore, we 2 autonomous signaling mechanism (i.e. it is an inherent prop- suggest that previous discrepancies in the assignment of erty of the mitochondrial ETC). Therefore, mtROS generation effects of inhibitors on ROS may be due to differences in should increase in hypoxia regardless of the experimental sys- experimental [O ]. Finally, the data set (see supplemental 2 tem being studied, including isolated mitochondria. In contrast material) may be useful in the mathematical modeling of to this hypothesis, we and others have demonstrated that ROS mitochondrial metabolism. generation by mitochondria is a positive function of [O ] across a wide range of values (0.1–1000 M O ) (15–18), suggesting that signaling mechanisms external to mitochondria may be required to facilitate the increased hypoxic mtROS production The production of reactive oxygen species (ROS) by mito- observed in cells. chondria has been implicated in numerous disease states, One limitation of our previous work (15) was that only a including but not limited to sepsis, solid state tumor survival, single respiratory condition was studied, namely succinate and diabetes (1). In addition, mitochondrial ROS (mtROS) play as respiratory substrate (feeding electrons into complex II) key roles in cell signaling (reviewed in Refs. 2 and 3). There exist plus rotenone to inhibit backflow of electrons through com- within mitochondria several sites for the generation of ROS, plex I (5, 7). The possibility exists that under different met- with the most widely studied being complexes I and III of the abolic conditions, which may lead to differential redox states electron transport chain (ETC). However, there is currently between the cytochromes in the ETC (19, 20), ROS genera- some debate regarding the relative contribution of these com- tion may exhibit a different response to [O ]. Thus, in the plexes to overall ROS production (4–9) and the factors that current study, we examined the response of mtROS genera- may alter this distribution. One such factor considered herein is tion to [O ] under 11 different conditions, using a variety of [O ]. Estimates of physiological [O ] within tissues (i.e. intersti- 2 2 respiratory substrates and inhibitors (for a thorough review tial [O ]) range from 37 down to 6 M at 5–40 m away from a of electron entry points to the ETC under various substrate/ inhibitor combinations, see Ref. 21). Fig. 1 shows a schematic □ S The on-line version of this article (available at http://www.jbc.org) contains of the mitochondrial ETC, highlighting sites of electron supplemental Tables S1–S3. entry resulting from various substrates, binding sites of To whom correspondence should be addressed: Box 604, Dept. of Anes- inhibitors, and major sites of ROS generation. Fig. 2 shows thesiology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-273-1626; Fax: 585-273-2652; E-mail: the specific details of each experimental condition, indicat- [email protected]. ing the predicted sites of ROS generation resulting from the The abbreviations used are: ROS, reactive oxygen species; mtROS, mito- use of each substrate/inhibitor combination. The legend to chondrial ROS; ETC, electron transport chain; ETFQOR, electron transfer flavoprotein quinone oxidoreductase. Fig. 2 provides an explanation of each condition. 16236 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 This is an Open Access article under the CC BY license. Mitochondrial ROS and O state 4 respiration. Where indi- cated, mitochondrial substrates and inhibitors were used at the fol- lowing concentrations: glutamate (10 mM), malate (5 mM), succinate (10 mM), palmitoyl-carnitine (1 M), rotenone (1 M), antimycin A (10 M), malonate (2 mM). They were present from the beginning of incubations before mitochondrial addition. Superoxide dismutase (80 units/ml) was present in all incuba- tions to ensure rapid dismutation of O to H O and to avoid scavenging 2 2 2 of the former by reaction with nitric oxide (NO ). This was a precaution, despite our previous observations FIGURE 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 that additional superoxide dis- complex I), succinate (which feeds electrons directly into complex II), and palmitoyl-carnitine (which feeds electrons mutase was not necessary in this into the ETC via acyl-CoA dehydrogenase as well as through the -oxidation pathway). (For a more thorough explanation, refer to Ref. 21.) Inhibitors used were rotenone (which inhibits at the downstream Q binding site of regard and that NO scavenging of complex I (9)), malonate (a competitive inhibitor of complex II (25, 26)), and antimycin A (a complex III inhibitor that O (which would lead to peroxyni- prevents electron flow to the Q site of complex III, thus stabilizing QH at the Q site (6, 28)). I O trite-mediated tyrosine nitration) was not occurring in hypoxia (15). The latter is also unlikely because the K for O of all NOS The results of these studies indicated that although ROS gener- m 2 ation under all experimental conditions exhibited the same overall isoforms is very high (6–24 M) (24), so NO generation actu- response to [O ](i.e. hyperbolic, with decreased ROS at low [O ]), ally decreases in hypoxia. Full details on each combination of 2 2 substrates/inhibitors and the putative sites of ROS generation the apparent K for O varied widely between metabolic states. m 2 resulting from each are given under “Results” and in the legends EXPERIMENTAL PROCEDURES to Figs. 1 and 2. All chemicals were the highest grade available from Sigma The steady-state [O ] reached in open flow respirometry is unless otherwise indicated. Male adult Sprague-Dawley rats not an independent variable but a result of the individual char- (250 g) were purchased from Harlan (Indianapolis, IN) and acteristics of each mitochondrial incubation. Therefore, it is were maintained in accordance with Ref. 53. All procedures not possible to use the raw data to calculate average rates of were approved by the University of Rochester Committee on ROS generation at a single [O ]. Thus, for each metabolic con- Animal Resources (protocol number 2007-087). Liver mito- dition, the empirical values of ROS generation across a range of chondria were isolated by differential centrifugation, as steady-state [O ] (typically 7–10 points/curve) were fitted to a described previously (15). single-substrate binding curve, employing Prism software Mitochondrial incubations were performed using an open (GraphPad, San Diego, CA), as described previously (15). The flow respirometry cell, as described previously (15, 22). Briefly, curve fit parameters (V , K ) were then used to extrapolate max m mitochondria were suspended in the liquid phase in a stirred ROS generation rates at common values of [O ], and these data chamber with a head space gas of tightly controlled pO flowing then averaged between individual experiments (n  5). above. Such a system, in which the liquid phase [O ] is meas- RESULTS ured with an O electrode, permits prolonged mitochondrial incubation at tightly controlled steady-state [O ] and the calcu- State 4 respiration rates (VO ; nmol of O /min/mg of pro- 2 2 2 lation of mitochondrial O consumption by a simplified Fick tein) under each metabolic condition were calculated across the equation (15, 22, 23). The O electrode was calibrated daily with range of [O ] values studied, as previously described (15, 22). 2 2 air-saturated deionized H O, with or without sodium dithion- The maximal VO (at high [O ]) for each condition is listed in 2 2 2 ite. The impact of additions to mitochondrial incubations (e.g. each panel of Fig. 2, whereas the full response curves of VO to substrates or inhibitors) on O solubility was no more than 0.4% [O ] are in Table S1.VO varied considerably between meta- 2 2 2 of the total. A fiber optic fluorimeter was built into the cham- bolic substrates. For example, a higher VO was observed with ber, permitting measurement of mitochondrial ROS generation complex II substrates (condition E) than with complex I sub- using the H O -sensitive dye Amplex red (23). Authentic H O strates (condition A). The consensus view is that because fewer 2 2 2 2 was added at the end of each experimental run to internally H are pumped across the inner membrane when electrons calibrate the fluorescent signal. Such a method ensures that the enter at complex II, the ETC has to work faster in condition E obtained signal truly reflects the net H O production and is (and thus consume more O ) to maintain the same H gradient 2 2 2 not affected by scavenging due to enzymes, such as catalase. as in condition A. Incubations were carried out in mitochondrial respiration The generation of ROS as a function of [O ] for metabolic buffer (15), with oligomycin (1 g/ml) present to enforce conditions A–L (Fig. 2) is illustrated in Fig. 3. Respiring on JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16237 Mitochondrial ROS and O complex I-linked substrates (glutamate plus malate in the pres- increased maximal ROS slightly (V 295) while causing a max ence of malonate to inhibit complex II), ROS generation was right shift in the curve (K  2.0 M O ; Fig. 3B). Similarly, m 2 maximal at 250 pmol/min/mg mitochondrial protein, whereas inhibition at complex III by antimycin A also increased ROS K was 0.25 M O (Fig. 3A). As expected, the addition of rote- (V 460) and further right-shifted the curve (K  5.0 M O ; m 2 max m 2 none, which inhibits at the downstream Q site of complex I, Fig. 3C). 16238 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mitochondrial ROS and O With succinate as the respiratory substrate, feeding electrons Despite the different sites of electron entry, all conditions into complex II (Fig. 3D), maximal ROS generation was 330 exhibited the same overall pattern of ROS generation in pmol/min/mg mitochondrial protein with a K of 1.8 M O . response to [O ], namely a hyperbolic function with lower ROS m 2 2 Some of this ROS was due to backflow of electrons through generation rate at lower [O ]. Thus, it appears that our previous complex I, since the addition of rotenone (Fig. 3E) brought the data set showing decreased mtROS at low [O ] (15) was not an V value down to 105 and the K to 0.7 M O . Similarly to artifact of the metabolic conditions chosen (succinate plus max m 2 the situation with complex I-linked substrates (see above), the rotenone). addition of antimycin A to succinate-respiring mitochondria Although information on ROS generation under different (Fig. 3F) raised maximal ROS generation to 420 pmol/min/mg substrate/inhibitor conditions is useful in the field of isolated mitochondrial protein and strongly right-shifted the curve mitochondrial bioenergetics, it would be more useful to know (K  12 M O ). Adding both rotenone and antimycin A m 2 the O sensitivity of ROS generation from putative sites within together (Fig. 3G) gave a V of 380 and a K of 4M O . Thus, max m 2 the ETC. Thus, a series of calculations was devised to estimate in both complex I- and II-linked respiration, antimycin A-in- ROS generation from each of four putative sites, at varying [O ] duced ROS generation is heavily O -dependent, having a much (Fig. 4). Below, the rationale behind each calculation is dis- greater K than base-line ROS generation (Fig. 3, C versus A, G cussed along with the results. versus E, and F versus D). Complex III Q Site—The rate of ROS generation from the Under conditions of dual electron entry at complexes I and II Q site of complex III was estimated by two methods. First, it (i.e. respiration on glutamate, malate, and succinate together) was estimated by using the rate of ROS generation obtained in (Fig. 3H), maximal ROS generation was 330 pmol/min/mg the presence of succinate as substrate (complex II) plus rote- mitochondrial protein, and K was 0.5 M O . As seen for com- m 2 none to inhibit electron backflow through complex I (i.e. con- plex I- or complex II-linked substrates alone, the addition of dition E) (5, 9, 27). Under this condition, ROS generation antimycin A to the dual electron entry condition (Fig. 3J) occurs primarily at the complex III Q site (6, 7, 28). Second, resulted in the highest ROS generation measured under any the rate of ROS generation due to backflow of electrons through condition (V  490) and a strongly right-shifted curve max complex I (calculated below) was subtracted from the rate of (K  9 M O ). m 2 ROS with succinate alone in which electrons flow both forward In mitochondria respiring on palmitoyl-carnitine, maximal through complex III and backward through complex I (condi- ROS was 290 pmol/min/mg mitochondrial protein, with a K tion D). The two values for complex III Q site ROS generation of 1.0 M O . Similar to the situation with complex II, some of were then averaged (Fig. 4A), resulting in a V of 150 and max this ROS may result from complex I backflow, since the addi- apparent K of 2.0. tion of rotenone resulted in a decrease in ROS (V  250) and max Complex I FMN Site—To estimate ROS generation by the a right shift in the curve (K  4 M O ). m 2 complex I FMN site, we used mitochondria respiring on com- DISCUSSION plex I-linked substrates alone (i.e. glutamate plus malate) in the presence of a complex II inhibitor to prevent electron entry due In the current study, we examined the response of mtROS to passage of substrates through the tricarboxylic acid cycle. generation to [O ] under 11 different conditions, using a variety The inhibitor chosen was malonate, since 2-thenoyltrifluoroac- of respiratory substrates and inhibitors (Fig. 2, A–L). Fig. 3 etone (29) exhibited an absorbance spectrum that interfered shows mtROS generation as a function of [O ] for each of the 11 with Amplex Red (not shown) and may also stimulate ROS conditions A–L. In conditions A–C, electrons entered the ETC generation at complex II (30, 31). Under condition A (gluta- at complex I, with complex II blocked by malonate (25, 26). In mate, malate, and malonate), some electron flux proceeds via conditions D–G, electrons entered at complex II. In conditions H and J, electrons entered at both complexes I and II, and in the Q pool to complex III. Thus, it is necessary to subtract ROS conditions K and L, electrons entered at the -oxidation elec- generation by the complex III Q site. Furthermore, it is insuf- tron transfer flavoprotein quinone oxidoreductase (ETFQOR). ficient to merely subtract ROS as calculated above, since that FIGURE 2. Pathways of electron flow for the substrate/inhibitor combinations used in conditions A–L. Each panel includes the respective maximal respiration rate (VO ; nmol of O /min/mg of protein) measured under each condition. A, glutamate/malate/malonate. Electrons enter through complex I, 2max 2 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 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 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 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 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 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 , 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 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 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 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 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 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. JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16239 Mitochondrial ROS and O FIGURE 3. O response of ROS generation rate for different substrate inhibitor combinations. ROS generation by isolated rat liver mitochondria under different steady state [O ] 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). 16240 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mitochondrial ROS and O FIGURE 3— continued flux was calculated from mitochondria respiring on succinate, ROS at O levels far below that at which the complex III Q site 2 O and the flux through the respiratory chain (i.e. VO ) was lower is already O -limited (K  2.0 M O ; see Fig. 4A). 2 2 m 2 with glutamate plus malate. This lower electron flux has two Notably, Fig. 5, which shows that the percentage of electron opposing effects on ROS generation by complex III Q . First, flux diverted to ROS increases as respiration slows down, might fewer electrons reach complex III, as shown by the VO in con- be misconstrued as demonstrating that mitochondrial ROS dition A (Fig. 2A), which was 39.3% of that in condition E (Fig. generation increases at low respiration rates (such as those 2E). Second, this slower electron flux through complex III caused by low [O ]). However, as we previously discussed (15), results in an increased dwell time for the ubisemiquinone rad- although a greater percentage of electrons may be diverted to ical at the Q site, which enhances ROS generation (32). To ROS, the absolute number of electrons flowing through the correct for this second effect, it is necessary to determine the respiratory chain and thus available for diversion decreases by a relationship between the percentage of electrons diverted to far greater magnitude, such that the absolute number of ROS ROS and the total electron flux (VO ). Fig. 5 shows this rela- generated is lower. From both ROS detection and cell signaling tionship for mitochondria respiring in condition E (succinate perspectives, the parameter that matters is not the percentage plus rotenone), indicating that a VO of 19.5 nmol of of electrons diverted to ROS but the absolute amount of ROS, O /min/mg results in 1.065% of electrons going to ROS. Low- which always decreases at low [O ]. 2 2 ering the VO to 7.7 (i.e. the VO in condition A) increases this Complex I Backflow—The backflow of electrons through 2 2 value to 1.817%. Thus, the percentage of electrons diverted to complex I causes ROS production at its downstream ubiqui- ROS is 1.71-fold greater in condition A versus condition E. none binding site (5, 9), which can be calculated in two ways. Combining these two correction factors (39.3%  1.71) indi- First, ROS generation in the presence of succinate alone (con- cates that it is necessary to subtract 67.2% of the ROS from the dition D) includes ROS from both forward and backward elec- complex III Q site (Fig. 4A) to reveal the residual ROS from tron flow. The contribution of backflow can be quantified by the complex I FMN site. The result is shown in Fig. 4B, and subtracting data obtained under the forward flow only condi- interestingly, the shape of the curve is not a classical hyperbolic tion (i.e. succinate plus rotenone, condition E). Second, palmi- function but instead indicates a V of 170 at 5 M O , with toyl-carnitine feeding electrons into the Q-pool can serve as an max 2 ROS declining very slightly at higher [O ]. The apparent K alternative source of electrons for complex I backflow. Similarly 2 m from this curve was estimated as 0.19 M O . These data there- to the succinate data (condition D versus E), the presence or fore suggest that the complex I FMN site is able to generate absence of rotenone to prevent backflow can also be applied to JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16241 Mitochondrial ROS and O 16242 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mitochondrial ROS and O complex III Q site (condition E) to reveal the residual ROS from the ETFQOR. Data from this calculation are shown in Fig. 4D, indicating a V of 200 and an apparent K of 5 M O . max m 2 Thus, although these data are consistent with the proposal that the ETFQOR can be a significant source of ROS (5), the high apparent K indicates that this may only occur at high [O ]. m 2 Dual Electron Entry—mtROS generation in the presence of both complex I- and II-linked substrates (condition H) was very high (V  330), with a K of 0.5 M O . As visualized in Fig. max m 2 4E, above 5 M O , the ROS generation was almost additive between the two individual substrate conditions (i.e. ROS with complex I substrates plus ROS with complex II substrates ROS with both complex I and II substrates). This suggests that at high [O ], some spare capacity exists in the ROS-generating FIGURE 5. ROS generation as a percentage of electron flux through the system, such that adding more electrons from either complex I respiratory chain, as a function of respiration rate (VO ). Two molecules of O are required to make one H O , so data on the y axis were calculated by or II can increase ROS generation. Thus, at high [O ], the mix of 2 2 2 2 dividing 2 ROS generation rate by the respiration rate. Data are from mito- substrates may profoundly impact on the rate of ROS genera- chondria respiring on succinate plus rotenone (condition E) and are taken tion. However, interestingly at lower [O ] (below 2 M; Fig. 4E, from the tables in the supplemental material. Error bars are eliminated for 2 clarity. The right-most point is state 4 respiration, with the remaining points on inset), the ROS generation rate under dual electron entry the curve originating from changes in VO due to titration of O levels. The 2 2 closely matched the rate with complex I substrates alone (i.e. arrows highlight specific values of VO referred to throughout and the extrap- adding electrons from complex II did not increase the rate of olated values of percentage of electrons diverted to ROS. ROS generation). This suggests that at low [O ], the generation palmitoyl-carnitine-linked ROS generation (i.e. condition K site for ROS (or something controlling it) may already be satu- versus L) to infer the rate of ROS from backflow. Averaged data rated, so adding more electrons cannot increase the rate any from these two calculations are shown in Fig. 4C, indicating a further. Therefore, in tissues, changes in the mix of substrates V of 135 and an apparent K of 0.9 M O . The occurrence (complex I versus complex II) may or may not have a differential max m 2 and physiological importance of ROS from complex I backflow effect on ROS generation, depending on the prevailing [O ]. remains unclear (5, 7, 9), and the data in Fig. 4C indicate the Notably, it has been observed that in skeletal muscle mito- importance of [O ] in regulating this phenomenon. In addition, chondria, ROS generation under dual electron entry far exceeds the ratio of complex I versus complex II substrates is expected the sum of rates with either complex I or complex II substrates to play an important regulatory role in vivo, since forward elec- alone (7). This may be due to different substrate preferences tron flow through complex I will effectively prohibit backflow between muscle and liver mitochondria, which may in turn be (21, 33, 34). related to differential expression levels of the various proteins ETFQOR—The ETFQOR of -oxidation is known to gener- of the oxidative phosphorylation machinery (35). Together, ate ROS (5). In the presence of palmitoyl-carnitine (condition these results highlight that patterns of ROS generation vary K) ROS generation is from three sites: the ETFQOR, complex greatly between different tissues and that tissue oxygenation is III Q site, and complex I backflow. To account for backflow, it another factor that may influence specific pathways of mito- is necessary to consider the rate of palmitoyl-carnitine-linked chondrial ROS generation. ROS generation in the presence of rotenone (condition L). To Effects of Inhibitors—The effect of mitochondrial inhibitors account for ROS from the complex III Q site under these on ROS generation has been widely studied, but the influence of conditions, a similar calculation is performed as above for the O on these effects has not been. In the current investigation, complex I FMN site (i.e. scaling of the complex III Q site data two inhibitors, rotenone and antimycin A, were examined. using a correction factor that considers the effects of respira- Rotenone binds at the downstream Q site within complex I, tion rate on ROS generation at this site). Electron flux through increasing ROS generation at the upstream FMN site. However, complex III in the presence of palmitoyl-carnitine plus rote- another effect of rotenone is to block electrons from exiting none (VO  2.8; Fig. 2L) is 14.3% of the flux with succinate plus complex I and proceeding via the Q pool to complex III (27). rotenone (VO  19.5; Fig. 2E). Furthermore, by reference to Thus, rotenone decreases ROS generation from the complex III Fig. 5, this lower electron flux results in a 2.37-fold increase in Q site. The effects of rotenone on overall ROS generation will the percentage of electrons donated to ROS (versus that manifest as a balance of these two effects, and herein we esti- observed at maximal flux). Combining these correction factors, mated that ROS generation at these two sites is differentially it is necessary to subtract 34% of the ROS generation from the O -sensitive (Fig. 4, A versus B). Comparing the rates of ROS FIGURE 4. Estimated O 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 [O ], as detailed under “Experimental Procedures” and calculated according to procedures outlined under “Discussion.” A, the Q site of complex III. B, the FMN site of complex I. C, the backflow of electrons through complex I. D, the ETFQOR of -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 [O ]from0to10 M O . For data values obstructed by the inset, see Fig. 3 (conditions A, E, and H). F, the effect of rotenone on the response 2 2 of mitochondrial ROS production to [O ](i.e. data from Fig. 3B minus Fig. 3A). G, the effect of antimycin A on the response of mitochondrial ROS production to [O ] when respiring on succinate (i.e. data are reproduced from Fig. 3, E (open symbols) and G (filled symbols)). JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16243 Mitochondrial ROS and O generation in mitochondria respiring on complex I-linked sub- oxidase (e.g. ischemia/hypoxia), compared with upstream com- strates in the presence (condition B) and absence (condition A) plexes that are relatively more “cushioned” from such events of rotenone (i.e. subtracting A from B) reveals an interesting (52). However, our results (Fig. 4) suggest that complex III is pattern for the effect of rotenone on ROS generation as a func- one of the least likely sites for ROS generation under hypoxic tion of [O ] (Fig. 4F). At high [O ], rotenone stimulates ROS conditions. Thus, we hypothesize that in hypoxia, ROS may 2 2 from the complex I FMN site, but it appears that rotenone- originate from an as yet unidentified mitochondrial source. induced inhibition of ROS from the complex III Q site is Alternatively, since the current experiments highlight that the insufficient to counteract this effect, and thus overall ROS gen- property of increased ROS in response to low O is not auton- eration increases. In contrast, at low [O ], although rotenone- omous to the mitochondrial respiratory chain, an additional stimulated ROS generation from the complex I FMN site still signal external to the mitochondrion may be required to facili- occurs, this is not enough to counteract the much larger tate a hypoxic increase in mtROS within cells. Such a signal is decrease in ROS from the complex III Q site, so the net effect probably absent from our isolated mitochondrial incubations. is an inhibition of ROS generation by rotenone. This effect may A third possibility is that differences in methodology (e.g. explain previous observations obtained by culturing cells at low choice of fluorescent ROS probes) and definitions of “hypoxia” [O ], in which rotenone inhibited ROS generation (36), com- may account for the varied reports of hypoxic ROS generation pared with cells or isolated mitochondria at high [O ], in which in the literature (for discussion, see Ref. 15). rotenone stimulated ROS generation (5, 9, 31, 37, 38). These In summary, the data presented herein suggest that overall results highlight the critical importance of [O ] as a variable ROS generation by isolated mitochondria under a variety of when using inhibitors to manipulate mitochondrial ROS. metabolic conditions decreases at low [O ]. Estimating the [O ] 2 2 The complex III inhibitor antimycin A has been widely used sensitivity of four putative ROS-generating sites within the to augment ROS generation from the complex III Q site (6, 28, ETC reveals a wide range of apparent K values. Thus, when O m 39), but the effect of physiological variations in [O ] on this considering the relative importance of these sites in contribut- phenomenon has not been studied. The effect of antimycin A ing to overall mtROS generation and redox balance, it is essen- on ROS generation as a function of [O ] is shown in Fig. 4G, tial to include [O ] as a variable. Finally, these data (see supple- 2 2 indicating that the -fold increase in ROS resulting from antimy- mental material) may facilitate the prediction of mtROS cin A addition is greater at high O levels (3-fold at 20 M O generation under a variety of metabolic conditions via incorpo- 2 2 versus 2-fold at 2 M O ). 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Cell Physiol 292, C698–707 tutes of Health, Bethesda, MD JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16245 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

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

Hoffman, David L.; Brookes, Paul S.
Journal of Biological Chemistry – Jun 1, 2009
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 24, pp. 16236 –16245, June 12, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species □ S Generation Depends on Metabolic Conditions Received for publication, December 18, 2008, and in revised form, March 18, 2009 Published, JBC Papers in Press, April 14, 2009, DOI 10.1074/jbc.M809512200 ‡ §1 David L. Hoffman and Paul S. Brookes ‡ § From the Departments of Biochemistry and Anesthesiology, University of Rochester Medical Center, Rochester, New York 14642 The mitochondrial generation of reactive oxygen species blood vessel (10). More recently, EPR oximetry has estimated (ROS) plays a central role in many cell signaling pathways, but tissue [O ] to be in the 12–60 M range (11). In addition, ele- debate still surrounds its regulation by factors, such as substrate gant studies with hepatocytes have shown that O gradients availability, [O ] and metabolic state. Previously, we showed exist within cells, such that an extracellular [O ]of6–10 M that in isolated mitochondria respiring on succinate, ROS gen- yields an [O ]of 5 M close to the plasma membrane, drop- eration was a hyperbolic function of [O ]. In the current study, ping to 1–2 M close to mitochondria deep within the cell (12). we used a wide variety of substrates and inhibitors to probe the In cardiomyocytes, at an extracellular [O ]of29 M, intracel- O sensitivity of mitochondrial ROS generation under different lular [O ] varied in the range 6–25 M (13). Clearly, different metabolic conditions. From such data, the apparent K for O of tissues consume O at different rates, so these gradients can m 2 putative ROS-generating sites within mitochondria was esti- vary considerably between tissue and cell types. mated as follows: 0.2, 0.9, 2.0, and 5.0 M O for the complex I By definition, the generation of reactive oxygen species by any flavin site, complex I electron backflow, complex III Q site, and mechanism, is an O -dependent process. However, measure- electron transfer flavoprotein quinone oxidoreductase of -ox- ments in intact cells have indicated that mtROS generation idation, respectively. Differential effects of respiratory inhibi- increases at lower O levels (1–5% O ) (14). Proponents of an 2 2 tors on ROS generation were also observed at varying [O ]. increase in mtROS in response to hypoxia suggest that under Based on these data, we hypothesize that at physiological [O ], such conditions, reduction of the ETC results in increased leak- complex I is a significant source of ROS, whereas the electron age of electrons to O at the Q site of complex III (14). Such a 2 O transfer flavoprotein quinone oxidoreductase may only con- model posits that increased hypoxic ROS is a mitochondria- tribute to ROS generation at very high [O ]. Furthermore, we 2 autonomous signaling mechanism (i.e. it is an inherent prop- suggest that previous discrepancies in the assignment of erty of the mitochondrial ETC). Therefore, mtROS generation effects of inhibitors on ROS may be due to differences in should increase in hypoxia regardless of the experimental sys- experimental [O ]. Finally, the data set (see supplemental 2 tem being studied, including isolated mitochondria. In contrast material) may be useful in the mathematical modeling of to this hypothesis, we and others have demonstrated that ROS mitochondrial metabolism. generation by mitochondria is a positive function of [O ] across a wide range of values (0.1–1000 M O ) (15–18), suggesting that signaling mechanisms external to mitochondria may be required to facilitate the increased hypoxic mtROS production The production of reactive oxygen species (ROS) by mito- observed in cells. chondria has been implicated in numerous disease states, One limitation of our previous work (15) was that only a including but not limited to sepsis, solid state tumor survival, single respiratory condition was studied, namely succinate and diabetes (1). In addition, mitochondrial ROS (mtROS) play as respiratory substrate (feeding electrons into complex II) key roles in cell signaling (reviewed in Refs. 2 and 3). There exist plus rotenone to inhibit backflow of electrons through com- within mitochondria several sites for the generation of ROS, plex I (5, 7). The possibility exists that under different met- with the most widely studied being complexes I and III of the abolic conditions, which may lead to differential redox states electron transport chain (ETC). However, there is currently between the cytochromes in the ETC (19, 20), ROS genera- some debate regarding the relative contribution of these com- tion may exhibit a different response to [O ]. Thus, in the plexes to overall ROS production (4–9) and the factors that current study, we examined the response of mtROS genera- may alter this distribution. One such factor considered herein is tion to [O ] under 11 different conditions, using a variety of [O ]. Estimates of physiological [O ] within tissues (i.e. intersti- 2 2 respiratory substrates and inhibitors (for a thorough review tial [O ]) range from 37 down to 6 M at 5–40 m away from a of electron entry points to the ETC under various substrate/ inhibitor combinations, see Ref. 21). Fig. 1 shows a schematic □ S The on-line version of this article (available at http://www.jbc.org) contains of the mitochondrial ETC, highlighting sites of electron supplemental Tables S1–S3. entry resulting from various substrates, binding sites of To whom correspondence should be addressed: Box 604, Dept. of Anes- inhibitors, and major sites of ROS generation. Fig. 2 shows thesiology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-273-1626; Fax: 585-273-2652; E-mail: the specific details of each experimental condition, indicat- [email protected]. ing the predicted sites of ROS generation resulting from the The abbreviations used are: ROS, reactive oxygen species; mtROS, mito- use of each substrate/inhibitor combination. The legend to chondrial ROS; ETC, electron transport chain; ETFQOR, electron transfer flavoprotein quinone oxidoreductase. Fig. 2 provides an explanation of each condition. 16236 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 This is an Open Access article under the CC BY license. Mitochondrial ROS and O state 4 respiration. Where indi- cated, mitochondrial substrates and inhibitors were used at the fol- lowing concentrations: glutamate (10 mM), malate (5 mM), succinate (10 mM), palmitoyl-carnitine (1 M), rotenone (1 M), antimycin A (10 M), malonate (2 mM). They were present from the beginning of incubations before mitochondrial addition. Superoxide dismutase (80 units/ml) was present in all incuba- tions to ensure rapid dismutation of O to H O and to avoid scavenging 2 2 2 of the former by reaction with nitric oxide (NO ). This was a precaution, despite our previous observations FIGURE 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 that additional superoxide dis- complex I), succinate (which feeds electrons directly into complex II), and palmitoyl-carnitine (which feeds electrons mutase was not necessary in this into the ETC via acyl-CoA dehydrogenase as well as through the -oxidation pathway). (For a more thorough explanation, refer to Ref. 21.) Inhibitors used were rotenone (which inhibits at the downstream Q binding site of regard and that NO scavenging of complex I (9)), malonate (a competitive inhibitor of complex II (25, 26)), and antimycin A (a complex III inhibitor that O (which would lead to peroxyni- prevents electron flow to the Q site of complex III, thus stabilizing QH at the Q site (6, 28)). I O trite-mediated tyrosine nitration) was not occurring in hypoxia (15). The latter is also unlikely because the K for O of all NOS The results of these studies indicated that although ROS gener- m 2 ation under all experimental conditions exhibited the same overall isoforms is very high (6–24 M) (24), so NO generation actu- response to [O ](i.e. hyperbolic, with decreased ROS at low [O ]), ally decreases in hypoxia. Full details on each combination of 2 2 substrates/inhibitors and the putative sites of ROS generation the apparent K for O varied widely between metabolic states. m 2 resulting from each are given under “Results” and in the legends EXPERIMENTAL PROCEDURES to Figs. 1 and 2. All chemicals were the highest grade available from Sigma The steady-state [O ] reached in open flow respirometry is unless otherwise indicated. Male adult Sprague-Dawley rats not an independent variable but a result of the individual char- (250 g) were purchased from Harlan (Indianapolis, IN) and acteristics of each mitochondrial incubation. Therefore, it is were maintained in accordance with Ref. 53. All procedures not possible to use the raw data to calculate average rates of were approved by the University of Rochester Committee on ROS generation at a single [O ]. Thus, for each metabolic con- Animal Resources (protocol number 2007-087). Liver mito- dition, the empirical values of ROS generation across a range of chondria were isolated by differential centrifugation, as steady-state [O ] (typically 7–10 points/curve) were fitted to a described previously (15). single-substrate binding curve, employing Prism software Mitochondrial incubations were performed using an open (GraphPad, San Diego, CA), as described previously (15). The flow respirometry cell, as described previously (15, 22). Briefly, curve fit parameters (V , K ) were then used to extrapolate max m mitochondria were suspended in the liquid phase in a stirred ROS generation rates at common values of [O ], and these data chamber with a head space gas of tightly controlled pO flowing then averaged between individual experiments (n  5). above. Such a system, in which the liquid phase [O ] is meas- RESULTS ured with an O electrode, permits prolonged mitochondrial incubation at tightly controlled steady-state [O ] and the calcu- State 4 respiration rates (VO ; nmol of O /min/mg of pro- 2 2 2 lation of mitochondrial O consumption by a simplified Fick tein) under each metabolic condition were calculated across the equation (15, 22, 23). The O electrode was calibrated daily with range of [O ] values studied, as previously described (15, 22). 2 2 air-saturated deionized H O, with or without sodium dithion- The maximal VO (at high [O ]) for each condition is listed in 2 2 2 ite. The impact of additions to mitochondrial incubations (e.g. each panel of Fig. 2, whereas the full response curves of VO to substrates or inhibitors) on O solubility was no more than 0.4% [O ] are in Table S1.VO varied considerably between meta- 2 2 2 of the total. A fiber optic fluorimeter was built into the cham- bolic substrates. For example, a higher VO was observed with ber, permitting measurement of mitochondrial ROS generation complex II substrates (condition E) than with complex I sub- using the H O -sensitive dye Amplex red (23). Authentic H O strates (condition A). The consensus view is that because fewer 2 2 2 2 was added at the end of each experimental run to internally H are pumped across the inner membrane when electrons calibrate the fluorescent signal. Such a method ensures that the enter at complex II, the ETC has to work faster in condition E obtained signal truly reflects the net H O production and is (and thus consume more O ) to maintain the same H gradient 2 2 2 not affected by scavenging due to enzymes, such as catalase. as in condition A. Incubations were carried out in mitochondrial respiration The generation of ROS as a function of [O ] for metabolic buffer (15), with oligomycin (1 g/ml) present to enforce conditions A–L (Fig. 2) is illustrated in Fig. 3. Respiring on JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16237 Mitochondrial ROS and O complex I-linked substrates (glutamate plus malate in the pres- increased maximal ROS slightly (V 295) while causing a max ence of malonate to inhibit complex II), ROS generation was right shift in the curve (K  2.0 M O ; Fig. 3B). Similarly, m 2 maximal at 250 pmol/min/mg mitochondrial protein, whereas inhibition at complex III by antimycin A also increased ROS K was 0.25 M O (Fig. 3A). As expected, the addition of rote- (V 460) and further right-shifted the curve (K  5.0 M O ; m 2 max m 2 none, which inhibits at the downstream Q site of complex I, Fig. 3C). 16238 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mitochondrial ROS and O With succinate as the respiratory substrate, feeding electrons Despite the different sites of electron entry, all conditions into complex II (Fig. 3D), maximal ROS generation was 330 exhibited the same overall pattern of ROS generation in pmol/min/mg mitochondrial protein with a K of 1.8 M O . response to [O ], namely a hyperbolic function with lower ROS m 2 2 Some of this ROS was due to backflow of electrons through generation rate at lower [O ]. Thus, it appears that our previous complex I, since the addition of rotenone (Fig. 3E) brought the data set showing decreased mtROS at low [O ] (15) was not an V value down to 105 and the K to 0.7 M O . Similarly to artifact of the metabolic conditions chosen (succinate plus max m 2 the situation with complex I-linked substrates (see above), the rotenone). addition of antimycin A to succinate-respiring mitochondria Although information on ROS generation under different (Fig. 3F) raised maximal ROS generation to 420 pmol/min/mg substrate/inhibitor conditions is useful in the field of isolated mitochondrial protein and strongly right-shifted the curve mitochondrial bioenergetics, it would be more useful to know (K  12 M O ). Adding both rotenone and antimycin A m 2 the O sensitivity of ROS generation from putative sites within together (Fig. 3G) gave a V of 380 and a K of 4M O . Thus, max m 2 the ETC. Thus, a series of calculations was devised to estimate in both complex I- and II-linked respiration, antimycin A-in- ROS generation from each of four putative sites, at varying [O ] duced ROS generation is heavily O -dependent, having a much (Fig. 4). Below, the rationale behind each calculation is dis- greater K than base-line ROS generation (Fig. 3, C versus A, G cussed along with the results. versus E, and F versus D). Complex III Q Site—The rate of ROS generation from the Under conditions of dual electron entry at complexes I and II Q site of complex III was estimated by two methods. First, it (i.e. respiration on glutamate, malate, and succinate together) was estimated by using the rate of ROS generation obtained in (Fig. 3H), maximal ROS generation was 330 pmol/min/mg the presence of succinate as substrate (complex II) plus rote- mitochondrial protein, and K was 0.5 M O . As seen for com- m 2 none to inhibit electron backflow through complex I (i.e. con- plex I- or complex II-linked substrates alone, the addition of dition E) (5, 9, 27). Under this condition, ROS generation antimycin A to the dual electron entry condition (Fig. 3J) occurs primarily at the complex III Q site (6, 7, 28). Second, resulted in the highest ROS generation measured under any the rate of ROS generation due to backflow of electrons through condition (V  490) and a strongly right-shifted curve max complex I (calculated below) was subtracted from the rate of (K  9 M O ). m 2 ROS with succinate alone in which electrons flow both forward In mitochondria respiring on palmitoyl-carnitine, maximal through complex III and backward through complex I (condi- ROS was 290 pmol/min/mg mitochondrial protein, with a K tion D). The two values for complex III Q site ROS generation of 1.0 M O . Similar to the situation with complex II, some of were then averaged (Fig. 4A), resulting in a V of 150 and max this ROS may result from complex I backflow, since the addi- apparent K of 2.0. tion of rotenone resulted in a decrease in ROS (V  250) and max Complex I FMN Site—To estimate ROS generation by the a right shift in the curve (K  4 M O ). m 2 complex I FMN site, we used mitochondria respiring on com- DISCUSSION plex I-linked substrates alone (i.e. glutamate plus malate) in the presence of a complex II inhibitor to prevent electron entry due In the current study, we examined the response of mtROS to passage of substrates through the tricarboxylic acid cycle. generation to [O ] under 11 different conditions, using a variety The inhibitor chosen was malonate, since 2-thenoyltrifluoroac- of respiratory substrates and inhibitors (Fig. 2, A–L). Fig. 3 etone (29) exhibited an absorbance spectrum that interfered shows mtROS generation as a function of [O ] for each of the 11 with Amplex Red (not shown) and may also stimulate ROS conditions A–L. In conditions A–C, electrons entered the ETC generation at complex II (30, 31). Under condition A (gluta- at complex I, with complex II blocked by malonate (25, 26). In mate, malate, and malonate), some electron flux proceeds via conditions D–G, electrons entered at complex II. In conditions H and J, electrons entered at both complexes I and II, and in the Q pool to complex III. Thus, it is necessary to subtract ROS conditions K and L, electrons entered at the -oxidation elec- generation by the complex III Q site. Furthermore, it is insuf- tron transfer flavoprotein quinone oxidoreductase (ETFQOR). ficient to merely subtract ROS as calculated above, since that FIGURE 2. Pathways of electron flow for the substrate/inhibitor combinations used in conditions A–L. Each panel includes the respective maximal respiration rate (VO ; nmol of O /min/mg of protein) measured under each condition. A, glutamate/malate/malonate. Electrons enter through complex I, 2max 2 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 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 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 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 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 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 , 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 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 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 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 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 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. JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16239 Mitochondrial ROS and O FIGURE 3. O response of ROS generation rate for different substrate inhibitor combinations. ROS generation by isolated rat liver mitochondria under different steady state [O ] 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). 16240 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mitochondrial ROS and O FIGURE 3— continued flux was calculated from mitochondria respiring on succinate, ROS at O levels far below that at which the complex III Q site 2 O and the flux through the respiratory chain (i.e. VO ) was lower is already O -limited (K  2.0 M O ; see Fig. 4A). 2 2 m 2 with glutamate plus malate. This lower electron flux has two Notably, Fig. 5, which shows that the percentage of electron opposing effects on ROS generation by complex III Q . First, flux diverted to ROS increases as respiration slows down, might fewer electrons reach complex III, as shown by the VO in con- be misconstrued as demonstrating that mitochondrial ROS dition A (Fig. 2A), which was 39.3% of that in condition E (Fig. generation increases at low respiration rates (such as those 2E). Second, this slower electron flux through complex III caused by low [O ]). However, as we previously discussed (15), results in an increased dwell time for the ubisemiquinone rad- although a greater percentage of electrons may be diverted to ical at the Q site, which enhances ROS generation (32). To ROS, the absolute number of electrons flowing through the correct for this second effect, it is necessary to determine the respiratory chain and thus available for diversion decreases by a relationship between the percentage of electrons diverted to far greater magnitude, such that the absolute number of ROS ROS and the total electron flux (VO ). Fig. 5 shows this rela- generated is lower. From both ROS detection and cell signaling tionship for mitochondria respiring in condition E (succinate perspectives, the parameter that matters is not the percentage plus rotenone), indicating that a VO of 19.5 nmol of of electrons diverted to ROS but the absolute amount of ROS, O /min/mg results in 1.065% of electrons going to ROS. Low- which always decreases at low [O ]. 2 2 ering the VO to 7.7 (i.e. the VO in condition A) increases this Complex I Backflow—The backflow of electrons through 2 2 value to 1.817%. Thus, the percentage of electrons diverted to complex I causes ROS production at its downstream ubiqui- ROS is 1.71-fold greater in condition A versus condition E. none binding site (5, 9), which can be calculated in two ways. Combining these two correction factors (39.3%  1.71) indi- First, ROS generation in the presence of succinate alone (con- cates that it is necessary to subtract 67.2% of the ROS from the dition D) includes ROS from both forward and backward elec- complex III Q site (Fig. 4A) to reveal the residual ROS from tron flow. The contribution of backflow can be quantified by the complex I FMN site. The result is shown in Fig. 4B, and subtracting data obtained under the forward flow only condi- interestingly, the shape of the curve is not a classical hyperbolic tion (i.e. succinate plus rotenone, condition E). Second, palmi- function but instead indicates a V of 170 at 5 M O , with toyl-carnitine feeding electrons into the Q-pool can serve as an max 2 ROS declining very slightly at higher [O ]. The apparent K alternative source of electrons for complex I backflow. Similarly 2 m from this curve was estimated as 0.19 M O . These data there- to the succinate data (condition D versus E), the presence or fore suggest that the complex I FMN site is able to generate absence of rotenone to prevent backflow can also be applied to JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16241 Mitochondrial ROS and O 16242 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mitochondrial ROS and O complex III Q site (condition E) to reveal the residual ROS from the ETFQOR. Data from this calculation are shown in Fig. 4D, indicating a V of 200 and an apparent K of 5 M O . max m 2 Thus, although these data are consistent with the proposal that the ETFQOR can be a significant source of ROS (5), the high apparent K indicates that this may only occur at high [O ]. m 2 Dual Electron Entry—mtROS generation in the presence of both complex I- and II-linked substrates (condition H) was very high (V  330), with a K of 0.5 M O . As visualized in Fig. max m 2 4E, above 5 M O , the ROS generation was almost additive between the two individual substrate conditions (i.e. ROS with complex I substrates plus ROS with complex II substrates ROS with both complex I and II substrates). This suggests that at high [O ], some spare capacity exists in the ROS-generating FIGURE 5. ROS generation as a percentage of electron flux through the system, such that adding more electrons from either complex I respiratory chain, as a function of respiration rate (VO ). Two molecules of O are required to make one H O , so data on the y axis were calculated by or II can increase ROS generation. Thus, at high [O ], the mix of 2 2 2 2 dividing 2 ROS generation rate by the respiration rate. Data are from mito- substrates may profoundly impact on the rate of ROS genera- chondria respiring on succinate plus rotenone (condition E) and are taken tion. However, interestingly at lower [O ] (below 2 M; Fig. 4E, from the tables in the supplemental material. Error bars are eliminated for 2 clarity. The right-most point is state 4 respiration, with the remaining points on inset), the ROS generation rate under dual electron entry the curve originating from changes in VO due to titration of O levels. The 2 2 closely matched the rate with complex I substrates alone (i.e. arrows highlight specific values of VO referred to throughout and the extrap- adding electrons from complex II did not increase the rate of olated values of percentage of electrons diverted to ROS. ROS generation). This suggests that at low [O ], the generation palmitoyl-carnitine-linked ROS generation (i.e. condition K site for ROS (or something controlling it) may already be satu- versus L) to infer the rate of ROS from backflow. Averaged data rated, so adding more electrons cannot increase the rate any from these two calculations are shown in Fig. 4C, indicating a further. Therefore, in tissues, changes in the mix of substrates V of 135 and an apparent K of 0.9 M O . The occurrence (complex I versus complex II) may or may not have a differential max m 2 and physiological importance of ROS from complex I backflow effect on ROS generation, depending on the prevailing [O ]. remains unclear (5, 7, 9), and the data in Fig. 4C indicate the Notably, it has been observed that in skeletal muscle mito- importance of [O ] in regulating this phenomenon. In addition, chondria, ROS generation under dual electron entry far exceeds the ratio of complex I versus complex II substrates is expected the sum of rates with either complex I or complex II substrates to play an important regulatory role in vivo, since forward elec- alone (7). This may be due to different substrate preferences tron flow through complex I will effectively prohibit backflow between muscle and liver mitochondria, which may in turn be (21, 33, 34). related to differential expression levels of the various proteins ETFQOR—The ETFQOR of -oxidation is known to gener- of the oxidative phosphorylation machinery (35). Together, ate ROS (5). In the presence of palmitoyl-carnitine (condition these results highlight that patterns of ROS generation vary K) ROS generation is from three sites: the ETFQOR, complex greatly between different tissues and that tissue oxygenation is III Q site, and complex I backflow. To account for backflow, it another factor that may influence specific pathways of mito- is necessary to consider the rate of palmitoyl-carnitine-linked chondrial ROS generation. ROS generation in the presence of rotenone (condition L). To Effects of Inhibitors—The effect of mitochondrial inhibitors account for ROS from the complex III Q site under these on ROS generation has been widely studied, but the influence of conditions, a similar calculation is performed as above for the O on these effects has not been. In the current investigation, complex I FMN site (i.e. scaling of the complex III Q site data two inhibitors, rotenone and antimycin A, were examined. using a correction factor that considers the effects of respira- Rotenone binds at the downstream Q site within complex I, tion rate on ROS generation at this site). Electron flux through increasing ROS generation at the upstream FMN site. However, complex III in the presence of palmitoyl-carnitine plus rote- another effect of rotenone is to block electrons from exiting none (VO  2.8; Fig. 2L) is 14.3% of the flux with succinate plus complex I and proceeding via the Q pool to complex III (27). rotenone (VO  19.5; Fig. 2E). Furthermore, by reference to Thus, rotenone decreases ROS generation from the complex III Fig. 5, this lower electron flux results in a 2.37-fold increase in Q site. The effects of rotenone on overall ROS generation will the percentage of electrons donated to ROS (versus that manifest as a balance of these two effects, and herein we esti- observed at maximal flux). Combining these correction factors, mated that ROS generation at these two sites is differentially it is necessary to subtract 34% of the ROS generation from the O -sensitive (Fig. 4, A versus B). Comparing the rates of ROS FIGURE 4. Estimated O 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 [O ], as detailed under “Experimental Procedures” and calculated according to procedures outlined under “Discussion.” A, the Q site of complex III. B, the FMN site of complex I. C, the backflow of electrons through complex I. D, the ETFQOR of -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 [O ]from0to10 M O . For data values obstructed by the inset, see Fig. 3 (conditions A, E, and H). F, the effect of rotenone on the response 2 2 of mitochondrial ROS production to [O ](i.e. data from Fig. 3B minus Fig. 3A). G, the effect of antimycin A on the response of mitochondrial ROS production to [O ] when respiring on succinate (i.e. data are reproduced from Fig. 3, E (open symbols) and G (filled symbols)). JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16243 Mitochondrial ROS and O generation in mitochondria respiring on complex I-linked sub- oxidase (e.g. ischemia/hypoxia), compared with upstream com- strates in the presence (condition B) and absence (condition A) plexes that are relatively more “cushioned” from such events of rotenone (i.e. subtracting A from B) reveals an interesting (52). However, our results (Fig. 4) suggest that complex III is pattern for the effect of rotenone on ROS generation as a func- one of the least likely sites for ROS generation under hypoxic tion of [O ] (Fig. 4F). At high [O ], rotenone stimulates ROS conditions. Thus, we hypothesize that in hypoxia, ROS may 2 2 from the complex I FMN site, but it appears that rotenone- originate from an as yet unidentified mitochondrial source. induced inhibition of ROS from the complex III Q site is Alternatively, since the current experiments highlight that the insufficient to counteract this effect, and thus overall ROS gen- property of increased ROS in response to low O is not auton- eration increases. In contrast, at low [O ], although rotenone- omous to the mitochondrial respiratory chain, an additional stimulated ROS generation from the complex I FMN site still signal external to the mitochondrion may be required to facili- occurs, this is not enough to counteract the much larger tate a hypoxic increase in mtROS within cells. Such a signal is decrease in ROS from the complex III Q site, so the net effect probably absent from our isolated mitochondrial incubations. is an inhibition of ROS generation by rotenone. This effect may A third possibility is that differences in methodology (e.g. explain previous observations obtained by culturing cells at low choice of fluorescent ROS probes) and definitions of “hypoxia” [O ], in which rotenone inhibited ROS generation (36), com- may account for the varied reports of hypoxic ROS generation pared with cells or isolated mitochondria at high [O ], in which in the literature (for discussion, see Ref. 15). rotenone stimulated ROS generation (5, 9, 31, 37, 38). These In summary, the data presented herein suggest that overall results highlight the critical importance of [O ] as a variable ROS generation by isolated mitochondria under a variety of when using inhibitors to manipulate mitochondrial ROS. metabolic conditions decreases at low [O ]. Estimating the [O ] 2 2 The complex III inhibitor antimycin A has been widely used sensitivity of four putative ROS-generating sites within the to augment ROS generation from the complex III Q site (6, 28, ETC reveals a wide range of apparent K values. Thus, when O m 39), but the effect of physiological variations in [O ] on this considering the relative importance of these sites in contribut- phenomenon has not been studied. The effect of antimycin A ing to overall mtROS generation and redox balance, it is essen- on ROS generation as a function of [O ] is shown in Fig. 4G, tial to include [O ] as a variable. Finally, these data (see supple- 2 2 indicating that the -fold increase in ROS resulting from antimy- mental material) may facilitate the prediction of mtROS cin A addition is greater at high O levels (3-fold at 20 M O generation under a variety of metabolic conditions via incorpo- 2 2 versus 2-fold at 2 M O ). 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