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Originally published In Press as doi:10.1074/jbc.M407715200 on August 17, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49064-49073, November 19, 2004
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Complex III Releases Superoxide to Both Sides of the Inner Mitochondrial Membrane*

Florian L. Muller{ddagger}§, Yuhong Liu{ddagger}, and Holly Van Remmen{ddagger}¶||

From the {ddagger}Departments of Cellular and Structural Biology and the Barshop Center for Longevity Studies, University of Texas Health Science Center and the ||South Texas Veterans Health Care System, San Antonio, Texas 78284-7762

Received for publication, July 9, 2004 , and in revised form, August 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for ~50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining ~50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mitochondrial electron transport chain is the main source of ATP in the mammalian cell and thus is essential for life (1). However, during energy transduction, a small number of electrons "leak" to oxygen prematurely (2–4), forming the oxygen free radical superoxide ( in its anionic form and in its protonated form), which has been implicated in the pathophysiology of a variety of diseases including Parkinson's, Huntington's, and Alzheimer's diseases as well as the aging process itself (5, 6). The detrimental significance of mitochondrial electron transport chain-derived superoxide is well illustrated by the lethal phenotype of mice lacking the mitochondrial matrix superoxide dismutase (Sod2) gene (7). While an increasing number of investigators have focused their attention on the potential pathological effects of mitochondrial superoxide (and its derivatives), there is a dearth of information on the mechanisms of deleterious superoxide formation by the electron transport chain. Indeed the state of knowledge has not changed much since the mid-1970s (4). As such, diminishing the rate of mitochondrial free radical production remains an elusive therapeutic strategy in the treatment of disease in which superoxide is thought to be involved. In this work, we seek to expand this area of investigation.

The basic facts of superoxide production can be summarized as follows. At the ultrastructural level, Complexes I and III are the main sites of mitochondrial superoxide production (2, 8, 9). In Complex I, the most likely sites of electron leakage are the iron-sulfur clusters (Refs. 8 and 10, although some evidence also points to the flavin (10)), while in Complex III, it is the Qo semiquinone (2, 3, 11–13). However, at the atomic level, the chemical details of either reaction remain completely unknown (for example, how does oxygen actually reach its reduction site?).

An important area of controversy, directly relevant to understanding the mechanism of superoxide formation, is to which side of the inner mitochondrial membrane either Complex I or Complex III releases superoxide (either to the mitochondrial matrix side or the cytoplasmic side (2)). Anionic superoxide (O2(aq)) is highly membrane-impermeable (14–17) such that biologically it is highly compartmentalized, i.e. there is no flux between the pools of matrix and cytoplasmic superoxide (16–19). The first studies on electron transport chain-derived superoxide production concluded that most superoxide must be extruded to the matrix side since superoxide was readily released from antimycin A- or rotenone-treated submitochondrial particles (SMPs,1 in which the matrix side faces the medium) but not from intact mitochondria (Refs. 20–23; for reviews, see Refs. 24 and 25). However, concomitant measurements of H2O2 production also revealed that only about half of the total electron leak in the presence of antimycin A (thus Complex III-derived (21, 24)) could be explained by net (outward) release from SMPs. The view that most superoxide production was directed toward the matrix was unchallenged until very recently when the x-ray structure of Complex III was solved (Ref. 26; for a review, see Ref. 27). F. L. M. pointed out that the x-ray structure of Complex III unambiguously shows the ubiquinol oxidation site (Qo site), the locus of superoxide production in Complex III (11), to be located immediately adjacent to the intermembrane space (IMS) and quite distant from the matrix (2). Based on this structural evidence, it was argued that some fraction of superoxide derived from the Qo site must be released to the IMS and that, if the data supporting superoxide release to the matrix were correct, it was the ability of Qo site-derived superoxide to reach the matrix that begged an explanation. Taking the latter data to be correct (21, 24, 28, 29), we formulated a simple model that could explain the release of Qo site superoxide to both sides of the inner mitochondrial membrane (2). Subsequently Han et al. (30) demonstrated that a Qo site-dependent DMPO signal could indeed be detected in mitoplasts, which these investigators interpreted as superoxide release to the IMS. Han et al. (30) were aware of the difficulties (2) regarding the reconciliation of the locus of superoxide production (the Qo site) with the release of superoxide to the matrix. Later St. Pierre et al. (31, 32) demonstrated that net extramitochondrial superoxide release could account for 25–75% of the total electron leak through Complex III. St. Pierre et al. (31, 32) argued that all Complex III-derived superoxide was released to the IMS and that the 75–25% unaccounted for could be explained by the action of IMS CuZn-SOD. In later work, Han et al. (33) took a similar position, and the view that Complex III exclusively releases superoxide to the cytoplasmic side of the inner membrane (i.e. IMS) seems to be embraced by the mainstream of the scientific community as judged by a (leading) recent textbook (Ref. 34, p. 128). The contradictory data from SMPs indicating that Complex III can release superoxide into the matrix were not discussed in Refs. 31, 33, and 34; however, it might be argued that SMPs are too disrupted to properly resolve the topology of the sites of superoxide production (SMPs can be contaminated with unsealed vesicles, rightside out vesicles, and sheets (35)). Alternatively, the technique used to reach the estimates of superoxide release in Refs. 31 and 33, and thereby the conclusion that all superoxide from Complex III is released into the IMS (31, 33, 34), is known to overestimate the flux of superoxide (36).

In the present report (preliminary results were presented at the 2003 Society for Free Radical Biology and Medicine conference (37)), we address previous experimental shortcomings and we challenge the newly popular opinion that Complex III only releases superoxide to the cytoplasmic side of the inner membrane. Using a refined version of the assay of St. Pierre et al. (31), we show that while superoxide derived from the Qo site of Complex III was indeed released from intact skeletal muscle mitochondria, this net extramitochondrial superoxide release accounts for no more than ~50% of H2O2 production (by extension ~50% of total superoxide production) from Complex III even in mitochondria lacking CuZn-SOD (from Sod1–/– mice (38)). To demonstrate that the remaining ~50% of superoxide is released into the matrix, we avoided the use of SMPs (noting the potentially confounding factors), instead using the aconitase inhibition assay (39) in intact mitochondria. Superoxide release by Complex III into the matrix was corroborated by the observation of a profound inhibition of aconitase activity, a sensitive target of superoxide exclusively located in that compartment. This inhibition of aconitase occurred even in the presence of exogenously added SOD and catalase, verifying that neither H2O2 nor re-entry of released superoxide could account for this effect. Our data are thus fully in agreement with initial conclusions from studies in SMPs (24), confirming that Complex III-derived superoxide can indeed reach the matrix. We discuss two models of superoxide production that explain this observation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Unless stated otherwise, all chemicals used in this study were obtained from Sigma.

Animals—All mice used in this study were in the C57B6/J background and housed in the vivarium of the Audie L. Murphy Veterans Affairs Hospital. CuZn-SOD knock-out (Sod1–/–) mice were generated by the laboratory of C. Epstein (38). Mice used in this study were between 3 and 8 months of age. Animals were anesthetized and sacrificed by cervical dislocation. All procedures were approved by the subcommittee for animal studies at the Audie L. Murphy Veterans Affairs Hospital.

Mitochondrial Purification—Mitochondria were purified from whole hind limb skeletal muscle (predominantly gastrocnemius and soleus) according to Chappell and Perry (40, 41). Hind limb skeletal muscle was excised, weighed, bathed in 150 mM KCl, and placed in Chappell-Perry buffer with nagarse. The minced skeletal muscle was homogenized with an all glass homogenizer. The homogenate was centrifuged for 10 min at 600 x g, and the supernatant was passed through two cheesecloth layers and centrifuged at 14,000 x g for 10 min. The resultant pellet was washed once in modified Chappell-Perry buffer with 0.5% bovine serum albumin and once in modified Chappell-Perry buffer without bovine serum albumin. Mitochondria were used immediately. Respiration was measured with a Clark electrode as described by Estabrook (42). The respiratory control ratio was ~5.5 with glutamate/malate and ~2.5 with succinate + rotenone, indicating intactness of the inner mitochondrial membrane. Protein concentration was measured with the Biuret method.

H2O2 Production—Mitochondrial H2O2 release was measured with the AmplexTM red-horseradish peroxidase method (Molecular Probes, Eugene, OR (43)). Horseradish peroxidase (HRP, 2 units/ml) catalyzes the H2O2-dependent oxidation of non-fluorescent Amplex red (80 µM) to fluorescent resorufin red (43). Since HRP is a large protein that does not cross membranes, this assay only detects H2O2 that has been released from the mitochondria (it cannot measure H2O2 inside mitochondria). 200 units/ml CuZn-SOD was added to convert all into H2O2, a necessity since reacts very rapidly with HRP and HRP-Compound I, resulting in underestimation of the actual rate of H2O2 production (36, 44). Fluorescence was followed at an excitation wavelength of 545 nm and an emission wavelength of 590 nm using a Fluoroskan Ascent type 374 multiwell plate reader (Labsystems, Helsinki, Finland). The slope of the increase in fluorescence is converted to the rate of H2O2 production with a standard curve. Addition of 450 units/ml catalase decreased this slope by ~99% (data not shown). We performed all assays at 30 °C in black 96-well plates. Substrates used were 9 mM succinate and 5 mM glutamate + malate. For each assay, one reaction well contained buffer only and another contained buffer with mitochondria to estimate the background oxidation rates of Amplex red and to estimate the rate of H2O2 release in mitochondria without substrate (state 1) (45). The reaction buffer consisted of 125 mM KCl, 10 mM HEPES, 5 mM MgCl2, 2mM K2HPO4, pH 7.44. In this .study we also used the Amplex red-H2O2 assay to estimate the rate of O2 production by measuring H2O2 production in the presence or absence of exogenously added SOD (31–33). A weakness of this approach is that superoxide production is overestimated because superoxide can both reduce and oxidize HRP (36, 44). To address this problem of the previous procedure (31–33), we added 20 µM acetylated cytochrome c (cyt c) to the Amplex red reaction buffer to act as a superoxide "sink" (instead of HRP). As such, in the absence of SOD, extramitochondrially released superoxide reduces acetylated cyt c (a reaction that does not yield H2O2 as a product). When SOD is added, extramitochondrial superoxide reacts with it, forming H2O2 instead. Thus, the increase in H2O2 formation upon SOD addition can be used to estimate net extramitochondrial superoxide release.

Superoxide Production—Superoxide production was measured by three direct methods: 1) MCLA (modified Cypridina luciferin analog) chemiluminescence, 2) dihydroethidine (DHE) fluorescence, and 3) inhibition of aconitase activity.

MCLA (2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-{alpha}]-pyrazin-3-one, Molecular Probes) is chemically very similar to coelenterazine (47) but exhibits brighter luminescence in response to superoxide. MCLA reversibly reacts with superoxide, forming an adduct whose irreversible decay generates light (~465 nm (48)). The apparent rate constant of this reaction is ~105 M–1 s–1 (48). Light emission was detected and quantified using a Fluoroskan-FL Ascent type 374 microplate luminometer (Labsystems) with opaque (white) 96-well plates. The photomultiplier was set to default with an integration time of 1000 ms. The MCLA signal was quantified as an integral of 20 s of continuous measurement and expressed as relative luminescence units/mg of mitochondrial protein. The reaction was conducted in 100 µl of reaction buffer containing ~0.5 mg/ml mitochondrial protein. The reaction buffer contained 125 mM KCl, 10 mM HEPES, 5 mM MgCl2, 2 mM K2HPO4, and 5 µM MCLA. As a positive control, we used the xanthine/xanthine oxidase system (49). 1 mM xanthine + 0.1 unit/ml xanthine oxidase caused a ~100-fold increase in chemiluminescent signal as compared with xanthine or xanthine oxidase alone, which did not differ significantly from reaction buffer alone. Addition of SOD (CuZn-SOD from erythrocytes) decreased the MCLA signal by over 98% (data not shown).

As a confirmation of the MCLA assay, we used dihydroethidine (Molecular Probes). This compound is oxidized by superoxide to a fluorescent product with an excitation maximum at 498 nm and an emission maximum at 598 nm (50). DHE (50 µM in the same buffer as described above) was tested with xanthine + xanthine oxidase. Confirming previous work (50), a steady increase in fluorescence could be detected for which the slope (the rate of superoxide formation) was reduced to zero by addition of 100 units/ml SOD (data not shown).

The third method of superoxide detection used in this work is the aconitase inhibition method developed by Gardner and Fridovich (29, 39, 51). Aconitase catalyzes the reversible isomerization of citrate to isocitrate (cis-aconitate being the intermediate). A low redox potential 4Fe-4S iron-sulfur cluster is required for this activity, and superoxide reacts with the latter at around ~106 M–1 s–1 (51, 52), inactivating the enzyme. While it is known that other reactive species, such as H2O2, can also inactivate aconitase, the reaction of aconitase with superoxide is several orders of magnitude faster than that with H2O2. Thus, aconitase activity is a sensitive index of superoxide levels both in vivo and in vitro (18, 28, 29, 53, 54). Aconitase is usually present in both the mitochondrial matrix and the cytoplasm (but encoded by two different genes resulting in different molecular weight proteins). In skeletal muscle, however, only mitochondrial matrix aconitase is present (aconitase 2), making this tissue ideal for probing the question of superoxide release directed specifically toward the matrix.

Aconitase activity is assayed (in detergent-dispersed samples) by measuring NADP+ reduction by citrate in the presence of isocitrate dehydrogenase (the conversion of citrate to isocitrate by aconitase being the rate-limiting step). We used a fluorometric method (excitation at 355 nm and emission at 460 nm) to quantify the reduction of NADP+. Mitochondria (~0.4 mg of protein/ml) were aliquoted in 96-well plates (100 µl of pH 7.44, 125 mM KCl, 10 mM HEPES, 5 mM MgCl2, 2 mM K2HPO4) and incubated at 30 °C up to 40 min as indicated. Substrates (9 mM succinate and 5 mM glutamate/malate) and inhibitors (10 µM antimycin A, 10 µM rotenone, 0.4 µM stigmatellin, and 0.4 µM myxothiazol) were added as indicated. For each experiment, 200 units/ml SOD and 900 units/ml catalase were also added to eliminate extramitochondrial H2O2 and superoxide. Incubation was stopped, and aconitase activity measurements were begun by the addition of 1 volume (100 µl) of 50 mM Tris, 0.6 mM MnCl2, 60 mM citrate, 0.2% Triton X-100, 100 µM NADP+, and 1 unit of isocitrate dehydrogenase (Sigma). Fluorometric measurements were then started immediately (Fluoroskan-FL Ascent type 374 microplate reader). The "blank" to measure aconitase-independent NADP+ reduction consisted of the same buffer except with isocitrate dehydrogenase omitted. The slope of the increase in NADPH fluorescence was taken as the amount of aconitase activity. Following previous work (29), the results were expressed as a percentage relative to control, which, in our case, consisted of untreated skeletal muscle mitochondria.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Complex I-derived Superoxide Is Vectorially Released into the Matrix and Does Not Escape from Intact Mitochondria—When mitochondria respire with an NADH-linked substrate (such as glutamate/malate), rotenone inhibition causes a profound increase in superoxide production (8, 55). It was reported that exogenous SOD addition does not further increase H2O2 release from rotenone-inhibited mitochondria (31). This observation was taken as evidence that no Complex I-derived superoxide was released from mitochondria and that Complex I releases superoxide exclusively into the matrix, although St. Pierre et al. (31) cautioned that this was by no means conclusive since dismutation of superoxide by CuZn-SOD (whether in the intermembrane space or as a cytoplasmic contaminant) could not be ruled out.

Here we expand on this observation using a refined technique and using mitochondria isolated from mice lacking CuZn-SOD. Addition of 200 units/ml exogenous SOD did not increase the rate of mitochondrial H2O2 release (as measured with Amplex red with 20 µM acetylated cyt c) in glutamate/malate + rotenone-treated mitochondria even when the mitochondria were derived from CuZn-SOD null (Sod1–/–) mice (Fig. 1A).



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  		FIG. 1.
Addition of SOD increases the Complex III-dependent rate of mitochondrial H2O2 release by ~2-fold but leaves that of Complex I unchanged. Mitochondria were incubated in Amplex red reaction buffer plus 20 µM acetylated cyt c with or without added SOD. The experiment was performed in mitochondria derived from mice lacking CuZn-SOD (Sod1–/–) as well as wild type (WT). The experiment was repeated three times (three wild type and three Sod1–/– mice) with S.E. shown. A, exogenous SOD addition (200 units/ml) did not lead to an increase in the rate of H2O2 release in mitochondria treated with 5 mM glutamate/malate 10 µM rotenone (Complex I-derived superoxide). Light gray bars, no SOD. Black bars, 200 units/ml exogenous SOD added. B, addition of SOD increased the rate of H2O2 release by 2-fold in mitochondria treated with 10 µM rotenone + 10 µM antimycin A and 9 mM succinate. Under such conditions, superoxide production from Complex III is maximal and that from other sources is negligible by comparison. White bars, no SOD. Dark gray bars, 200 units/ml exogenous SOD added. Net extramitochondrial release of superoxide thus accounts for approximately half of the total H2O2 produced by Complex III but none of that produced by Complex I. prot., protein.

 
Since this indirect method is not suited to detecting low levels of superoxide, we wished to follow up on this observation by measuring superoxide release directly. Superoxide release was measured chemiluminescently with 5 µM MCLA on the same skeletal muscle mitochondria preparations that were used above for H2O2 assays. For comparative purposes, the data in Fig. 2 were normalized with respect to oxidant production in state 1 (i.e. mitochondria without substrate (45)). Addition of glutamate/malate increased the MCLA signal by around ~2-fold compared with mitochondria alone (state 1), but addition of rotenone with glutamate/malate did not result in any further increase in MCLA chemiluminescence as compared with glutamate/malate alone (Fig. 2). Yet rotenone + glutamate/malate drastically increased H2O2 release (measured with Amplex red), indicating that while superoxide was not being released from, it was still being produced in mitochondria, most likely being released into the matrix. In corroboration of this idea, a strong MCLA signal was observed in inverted SMPs (in which the matrix side faces the outside medium) in response to treatment with 100 µM NADH + rotenone (data not shown). This result was reported several times previously using different methods (22, 23) and indicates that superoxide produced by Complex I under rotenone inhibition is released into the matrix. To prove that this is also true in intact mitochondria, we measured inhibition of mitochondrial matrix aconitase activity, a sensitive target of superoxide. Addition of 10 µM rotenone to mitochondria respiring on 5 mM glutamate/malate resulted in a profound inhibition of aconitase activity (Fig. 3) even when exogenous catalase and SOD were present. Thus, using both indirect (H2O2 with Amplex red/cyt c± SOD) and direct methods (MCLA and aconitase), we demonstrated that, although Complex I produces large quantities of , no detectable levels escape from intact mitochondria.



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  		FIG. 2.
Both antimycin A and rotenone stimulate mitochondrial H2O2 release, but only antimycin A promotes extramitochondrial superoxide release. Skeletal muscle mitochondria (~0.4 mg/ml) were incubated in either Amplex red reaction buffer (with SOD) or with 5 µM MCLA in 125 mM KCl, 5 mM HEPES, 2 mM K2HPO4. For comparative purposes, rates of superoxide release with MCLA and H2O2 release with Amplex red were expressed relative to state 1 (mitochondria without added substrate), which was defined as `100.`As reported many times before, antimycin A + succinate as well as rotenone + glutamate/malate yielded a drastic increase in H2O2 release (white bars). However, only antimycin A + succinate (hence superoxide from Complex III) caused an increase in the MCLA signal (superoxide release, gray bars). The MCLA superoxide release with rotenone + glutamate/malate was not different from glutamate/malate alone. The MCLA chemiluminescent signal was insensitive to added catalase but was completely eliminated by the exogenous addition of 200 units/ml SOD (black bars). Each experiment was repeated at least three times with S.E. shown. Essentially the same result (no increase in superoxide release by addition of rotenone to glutamate/malate-respiring mitochondria) was obtained using DHE fluorescence to measure superoxide release (data not shown). We thus confirmed the conclusions reached from Fig. 1: 1) while Complex I produces large amounts of superoxide, none escapes from intact mitochondria, and 2) contrary to Complex I, Complex III readily releases superoxide from intact mitochondria. succ, succinate; G/M, glutamate/malate.

 



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  		FIG. 3.
Both Complexes I and III release superoxide into the mitochondrial matrix. Mitochondria (~0.4 mg/ml) were incubated with substrate (5 mM glutamate/malate) and 10 µM rotenone (dark gray bar) or 10 µM antimycin A (shaded bar). 900 units/ml catalase and 200 units/ml SOD were also added to prevent extramitochondrial or H2O2 from playing any part in the observed aconitase inhibition. Aconitase activity was measured 17 min after the incubation of mitochondria with substrates (and inhibitors) was begun. Following previous work (29), results were expressed as a percentage of initial (before incubation) aconitase activity. The experiment was repeated three times with S.E. shown.

 
Extramitochondrial Superoxide Release Accounts for No More than 50% of Total Electron Leak at Complex III—Next we addressed the sidedness of superoxide release from the Qo site of Complex III. In Fig. 2, data show that, in contrast to the effects of rotenone, antimycin A readily induced a large MCLA chemiluminescent signal in intact mitochondria that was completely eliminated by addition of exogenous SOD. These data thus indicate that Complex III can release superoxide to the cytoplasmic side of the inner mitochondrial membrane (i.e. into the IMS). We sought to quantify what fraction of Complex III-derived superoxide could be accounted for by this extramitochondrial superoxide release.

In Fig. 1, we present data addressing this question. We improved upon the methodology of previous investigators (Refs. 31 and 33 and see "Experimental Procedures") and performed the assays in mitochondria from mice lacking CuZn-SOD (38). In mitochondria treated with rotenone and antimycin A and supplemented with succinate, addition of SOD increased H2O2 production ~2-fold, indicating that ~50% of total H2O2 generated by Complex III can be explained by direct extramitochondrial release of superoxide. This leaves ~50% of total electron leak unaccounted for, yet our experiments conducted with Sod1–/– mitochondria ruled out dismutation of superoxide in the IMS. We hypothesize that the remaining 50% of H2O2 release resulted from superoxide released into the matrix (and subsequently dismutated by Mn-SOD).

Complex III-derived Superoxide Reaches the Mitochondrial Matrix—Pioneering data using SMPs suggested that Complex III-derived superoxide can reach the mitochondrial matrix (20, 21, 24). In the present study we revisited this question in intact mitochondria using aconitase inactivation to measure superoxide release into the mitochondrial matrix.

In Fig. 4, the effect of antimycin A on aconitase activity in succinate-supplemented mitochondria is shown. After a 40-min incubation, aconitase activity was decreased by about 95% as compared with succinate only-treated mitochondria. To eliminate the possibility of Complex I-derived superoxide production due to reverse electron flow, we performed the same experiments with added rotenone. These experiments were conducted with 900 units/ml catalase and 200 units/ml superoxide dismutase present to rule out that H2O2 or re-entry of superoxide was responsible for aconitase inactivation (Figs. 4 and 5). Next we show that the antimycin A-dependent inhibition of aconitase was largely prevented by addition of 0.4 µM stigmatellin (Fig. 5), which binds to the distal Qo site and prevents superoxide formation at Complex III (12, 27, 56, 57). Although we cannot rule out completely that superoxide derived from other sources may play a role in the inhibition of aconitase brought on by antimycin A, the fact that this can be largely prevented by stigmatellin indicates that the majority of aconitase inhibition was solely due to superoxide derived from the Qo site. Finally we repeated the experiments in Fig. 5 at pH 8.75 and 6.75 and in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These conditions did not change the results obtained in Fig. 5 (data not shown). We conclude from these data that superoxide derived from the Qo site does, in fact, reach the matrix by a pathway that is independent of bulk pH and proton gradient.



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  		FIG. 4.
Antimycin A treatment drastically inhibits mitochondrial matrix aconitase activity even in the presence of extramitochondrial SOD and catalase. ~0.4 mg/ml mitochondrial protein was incubated at 30 °C in the presence of 200 units/ml SOD, 900 units/ml catalase, 9 mM succinate, 10 µM antimycin A, and 10 µM rotenone as indicated. Rotenone was added to prevent reverse electron transfer through Complex I. The results were expressed as a percentage of initial aconitase activity (untreated mitochondria). The fact that antimycin A was able to inhibit aconitase activity despite the presence of exogenous SOD and catalase can only be explained by superoxide from Complex III released directly into the matrix. The reaction was stopped with the addition of aconitase reaction buffer (see "Experimental Procedures") at 0 (white bars), 10 (light gray bars), 20 (dark gray bars), and 40 min (black bars). The experiment was repeated at least four times with S.E. shown.

 



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  		FIG. 5.
Stigmatellin, which blocks superoxide production at the Qo site (13, 37, 57), prevents matrix aconitase inactivation. Mitochondria were treated with 10 µM rotenone (to prevent electron backup through Complex I) and 9 mM succinate (control) as well as 10 µM antimycin A and 0.4 µM stigmatellin as indicated. The inhibitors were preincubated with mitochondria for 5 min before succinate addition (to allow equilibration). The reactions (with added SOD and catalase) were then incubated at 30 °C for 20 min at which time aconitase activity was measured. Each bar represents the average of three experiments with S.E. shown. Antimycin A caused a substantial inhibition of aconitase activity, indicating superoxide release into the matrix. Concomitant addition of stigmatellin with antimycin A largely prevented this inhibition (very similar results were obtained with myxothiazol, data not shown). Stigmatellin is a Qo site inhibitor known to block antimycin A-induced superoxide formation from Complex III (37, 56, 57). Stigmatellin prevents superoxide formation by sterically blocking ubiquinol binding and formation of the Qo semiquinone (12, 27). Taken together, these data indicate that superoxide derived from Complex III was able to reach the mitochondrial matrix and that this superoxide originated from the Qo site semiquinone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Vectorial Release of Complex I-derived Superoxide into the Matrix—It is generally accepted that does not cross membranes. Empirical evidence backs up this view (14–16). The reason for this impermeability is the strong negative charge of , which is the predominant form of superoxide in aqueous solution at physiological pH (16, 44). The protonated form of superoxide, (pKa, 4.9), is readily membrane-permeable but is only present in very low amounts at physiological pH (less than 0.2% at pH 7.44 (44)). Thus, in the cell each membrane-bound compartment has its own SOD isozyme, and overexpression of one will not compensate for the loss of another SOD isozyme in a different compartment (18, 19). Given the fact that superoxide is very selective in its targets (58) and that once released into a membrane-bound compartment it cannot escape, understanding the sidedness of superoxide release by its sites of generation is essential to clarify the role of mitochondrial superoxide production in pathophysiology.

In rotenone-inhibited Complex I (in the presence of NADH-linked substrates), the oxygen reduction site (or locus of superoxide generation) is somewhere between the flavin (FMN) and the rotenone binding sites (8, 23, 59). Since the x-ray structure of Complex I has not yet been elucidated, the exact position of the rotenone binding site remains unknown. However, the rough outline of Complex I is known from cryoelectron microscopy (60, 61); the approximate loci of the rotenone and the flavin binding sites are known from biochemical and mutagenic studies (62). Finally it is also known that iron-sulfur cluster-modifying agents (chloromercury benzoate and ethoxyformic anhydride) profoundly decrease reactive oxygen species production elicited by rotenone with NADH-linked substrates (8). Thus, the site of superoxide formation must be somewhere on the hydrophilic fraction, which consists of the matrix-protruding arm of Complex I (Fig. 6). One would then hypothesize that superoxide produced from this locus should be exclusively released into the mitochondrial matrix. The data presented in this study support this hypothesis.



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  		FIG. 6.
The exclusive release of Complex I-derived superoxide into the mitochondrial matrix is in agreement with the hypothesis that the iron-sulfur clusters are the locus of oxygen reduction (8). Cryoelectron microscopy studies have delineated a "shoelike" general shape of Complex I (61). EPR as well as site-directed mutagenesis studies place the iron-sulfur clusters in the long hydrophilic, matrix-protruding arm (62). The rotenone binding site is located at the hydrophobic/hydrophilic interface (62). Our present data indicate that, in the presence of rotenone, Complex I releases superoxide exclusively into the mitochondrial matrix. This result is exactly what one would expect from the current notion that it is the hydrophilic arm iron-sulfur clusters that are the source of superoxide (8). UQH2, ubiquinol.

 
Using direct methods of superoxide detection (MCLA and DHE), we failed to observe a significant increase in superoxide release from intact skeletal muscle mitochondria (Figs. 1 and 2) in response to the addition of rotenone with glutamate/malate (an NADH-linked electron source). Yet the same treatment, as was reported many times before (8, 55), drastically increased H2O2 production (indicating that superoxide was being produced in, but not released out from, intact mitochondria). Rotenone + glutamate/malate led to a profound inhibition of aconitase activity in intact mitochondria, indicating that superoxide was being released into the matrix (Fig. 3). The question why MCLA (and DHE) would not react with matrix superoxide inevitably arises, since these probes are relatively large hydrophobic molecules that readily permeate cells, thus readily entering into the mitochondrial matrix. The simple answer is that both probes probably reach the matrix but are vastly out-competed (for reacting with superoxide) by the ~10 µM Mn-SOD in that compartment (Table I). Based on the kinetic data in Table I, we speculate that the methodology used by Han et al. (Ref. 33, using the spin trap DMPO) should yield results very similar to those obtained here with MCLA: these probes should detect superoxide released extramitochondrially but not superoxide in the mitochondrial matrix.


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  		TABLE I
MN-SOD vastly outcompetes superoxide-measuring probes in the matrix

 
The indirect method of superoxide measurement (Amplex red-H2O2 assay plus acetylated cyt c with or without added SOD) also failed to show any increase in extramitochondrial release in response to rotenone + glutamate/malate, confirming the aforementioned results. Since more or less identical results were obtained with mitochondria from mice lacking CuZn-SOD (which is present in the intermembrane space and the cytoplasm (63)), we may conclude that, despite high levels of Complex I superoxide generation toward the matrix, no detectable levels escape from intact mitochondria.

This finding strongly supports the hypothesis that the locus of superoxide production in Complex I is somewhere on the hydrophilic, mitochondrial matrix-protruding arm (Fig. 6). We suggest that the simplest mechanism for superoxide production at this site is a single electron transfer to O2 in the aqueous phase. An additional implication from our results is that the extramitochondrial superoxide release in the absence of respiratory inhibitors (Fig. 2, "succinate" and "Glutamate/malate") cannot be due to Complex I, and we posit that it must have originated from Complex III.

Complex III-derived Superoxide Is Released to Both the Matrix and the Intermembrane Space—Much more is known about the structure (several independent x-ray structures now published (26, 64, 65)) and mechanism of proton pumping (the modified Q cycle (27)) of Complex III than of Complex I. Yet despite 30 years of research, the mechanism of superoxide production at Complex III remains incompletely understood. In brief summary, Complex III superoxide production was first observed as H2O2 released by mitochondria in response to antimycin A (21, 66, 67) that was subsequently (in experiments with SMPs) demonstrated to have originated as the stoichiometric dismutation product of (21, 68). The experiments with SMPs also led to the long held conclusion that Complex III-derived superoxide must be released exclusively to the mitochondrial matrix (for a review, see Ref. 24) because SMPs are inverted (meaning the matrix side faces the medium). Subsequently Turrens et al. (11) determined that the Qo semiquinone, rather than cytochrome bL (for a detailed review of the structure and function of Complex III, see Ref. 27), was the source of superoxide under antimycin A treatment. More recently, the resolution of the x-ray structure demonstrated unambiguously that the Qo site is immediately adjacent to the intermembrane space (Fig. 7). The close proximity of the Qo site to the IMS previously led us to conclude that a fraction of Complex III-derived superoxide must invariably be released to the cytoplasmic side of the inner membrane (2). This was experimentally demonstrated shortly afterward (30). The position of the Qo site in the x-ray structure of Complex III also made the possibility of superoxide release into the matrix much more problematic, considering the high membrane impermeability of anionic superoxide (14–16); indeed most investigators have taken the position that Complex III only releases superoxide to the cytoplasmic side of the inner mitochondrial membrane (31, 33), in disagreement with the initial work in SMPs (21, 68). Despite some in vivo evidence (29), data from SMPs could be explained away as an artifact of preparation (for example, incompletely inverted SMPs, sheets, or other membrane fragments (35)). We believe, however, the SMP data to be valid and that an objective reading of the literature circumstantially supports that Complex III releases superoxide to both sides of the inner mitochondrial membrane (2). In this work, we provide direct supporting evidence for this in intact mitochondria.



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  		FIG. 7.
The x-ray structure of Complex III unambiguously shows that the Qo site (occupied by the Qo site inhibitor, stigmatellin in green) is located immediately next to the intermembrane space. This view was generated from the coordinates of the structure of Zhang et al. (Protein Data Bank code 3BCC [PDB] (26)) using Weblab Viewer Pro software. The orange lines indicate the limits of the lipid bilayer of the inner mitochondrial membrane. The functional complex consists of a dimer of 11 subunits each; for simplicity only a monomer of the three catalytically active subunits is shown. Cytochrome b is in red, cytochrome c1 is in blue, and the Rieske iron-sulfur protein is in yellow. To the right is a schematic representation of the structure with the major cofactors and ubiquinone binding sites indicated. The ubiquinol oxidation site (Qo site), the locus of superoxide formation (11), is formed mostly by cytochrome b and covered by the Rieske iron-sulfur protein. In this x-ray structure, it is occupied by stigmatellin (26). Structures in which this site is occupied by myxothiazol and other Qo site inhibitors have also been published (27). For a detailed review on the structure and mechanism of Complex III, see Ref. 27.

 
St. Pierre et al. (31, 32) and Han et al. (33) reported that up to 70% of the total H2O2 release can be accounted for by net extramitochondrial superoxide release (31–33). However, the methodology used to estimate the flux of superoxide in previous work relied on HRP as the superoxide sink (which is known to cause overestimation of the superoxide flux (36), see "Experimental Procedures"). We modified the methodology taking the above into consideration. First, we used lower levels of HRP (1 unit of HRP/ml for the Amplex red assay used here versus >10 units/ml in Refs. 31 and 33). Second, we added 20 µM acetylated cyt c as a superoxide sink. As shown in Fig. 1, ~35% of the total H2O2 production from Complex III (succinate + antimycin A) was due to net extramitochondrial superoxide release in wild-type mouse skeletal muscle mitochondria. As such, our data are about equal to the lower range of values of previous work (31–33). To address whether the remaining fraction of H2O2 release was simply due to dismutation catalyzed by CuZn-SOD (as was suggested by St. Pierre et al. (31)), we repeated the above experiments in skeletal muscle mitochondria from Sod1–/– mice (which lack CuZn-SOD). We found that in Sod1–/– mitochondria ~50% of total H2O2 flux (versus ~35% in wild-type mitochondria) can be ascribed to direct extramitochondrial release, leaving ~50% unaccounted for.

We hypothesized that the fraction of H2O2 production that could not be accounted for by extramitochondrial release was due to superoxide released into the matrix. To test this idea further, we measured the activity of matrix aconitase, an enzyme that is selectively inactivated by superoxide (39, 51). Our data in Fig. 5 show that antimycin A treatment caused a substantial inhibition of aconitase activity and that this could be largely prevented by the concomitant addition of the Qo site inhibitor stigmatellin. We performed the experiment in the presence of excess catalase and SOD: this ensured that neither H2O2 nor extramitochondrial was responsible for the inactivation of aconitase. But inhibition of aconitase occurred despite the presence of added SOD and catalase. The simplest explanation for this observation is that Complex III Qo site-derived superoxide is released into the matrix by a pathway that is independent of the bulk aqueous phase. We also varied the pH of the medium from pH 6.70 to pH 8.75. The results at these two extreme pH conditions were essentially the same as what we observed at pH 7.44 (data not shown). Knowing the pK of to be 4.9, only ~1% of aqueous superoxide would be in the form at neutral pH (69). We thus conclude that superoxide derived from the Qo site can indeed reach the matrix, independent of bulk pH, in agreement with previous work in SMPs and in vivo (29).

Two Models That Can Explain the Release of Complex III-derived Superoxide to Both Sides of the Inner Membrane—As we pointed out in the Introduction, the ability of membrane-impermeable superoxide to reach the matrix requires an explanation. We have previously proposed two alternative hypotheses, outlined in Fig. 8, to explain this seemingly contradictory observation. First, let us state that the simplest model for superoxide production, a one-electron transfer from ubisemiquinone to O2, resulting in formation in the aqueous phase (i.e. IMS) cannot adequately explain this observation (we have discussed this in detail previously (2)). To explain that Qo site-derived superoxide reaches both sides of the inner mitochondrial membrane the model requires impermeable to somehow cross the inner mitochondrial membrane. This could not be accomplished through anion channels (since would have to move against the membrane potential) even if hypothetical anion channels capable of channeling superoxide did exist. Liu (70) suggested that aqueous in the IMS could be protonated to , which could then diffuse through the inner membrane. This model cannot explain our present findings since neither raising the pH to 8.75 nor adding an uncoupler (both treatments drastically decrease the concentration of aqueous protons in the IMS) attenuated the inactivation of matrix aconitase (data not shown).



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  		FIG. 8.
Two models to explain how Complex III releases superoxide to both sides of the inner mitochondrial membrane. We propose two models to explain how superoxide derived from the Qo site can reach both sides of the inner mitochondrial membrane. In the first mechanism (shown in gray arrows), a neutral ubisemiquinone () could diffuse out of the Qo site along the tunnel that (the x-ray structure shows) connects the Qo site to the bulk hydrophobic lipid phase. At the lipid/aqueous phase interface, the ubisemiquinone would deprotonate (the pKa is considerably lower in the aqueous phase as compared with aprotic solvents (27)) and react with oxygen to form aqueously solvated superoxide (). The second model (shown in dark red arrows) argues that superoxide is formed in the hydrophobic part of the Q site via a proton-coupled electron transfer from a neutral ubisemiquinone. For thermodynamic reasons, only not can be formed in non-aqueous environments (74). Once formed, would diffuse into lipid phase through the same pathway as in the first model and would ultimately result in the release of at both sides of the inner mitochondrial membrane. For a more detailed discussion on the kinetic possibilities of this model, see Refs. 2 and 13.

 
But we argue that two previously proposed hypotheses can accommodate these new data. Our first hypothesis (13) posits that a neutral (uncharged) semiquinone escapes from the Qo site through the hydrophobic tunnel that connects the Qo site to the membrane lipid phase and opens up roughly in the middle of the inner mitochondrial membrane (see Fig. 8; for a detailed discussion on the structure of Complex III, see Refs. 2 and 71). From there, the neutral semiquinone would be able to diffuse to both sides of the inner membrane with more or less equal likelihood. At the lipid/aqueous phase interface, the semiquinone would release its proton and react with O2, forming aqueous, anionic . Equal propensity of the semiquinone to diffuse to either side of the membrane would explain why superoxide release to both sides of the inner mitochondrial membrane is observed. How well does such a scenario fit with the current understanding of the catalytic mechanism of Complex III? The ability of the semiquinone to escape from the Qo site would be a natural consequence of the Q cycle in its current form (27, 72, 73) since the Q cycle proposes a very thermodynamically unstable, hence very loosely bound, Qo semiquinone (72).

Our second hypothesis (2) states that oxygen reacts with a neutral semiquinone in the hydrophobic part of the Qo site to form . At first sight, this hypothesis would seem very similar to the proposal of Liu (70) thus suffering the same drawbacks. However, the critical difference between these models is that our hypothesis has forming in the hydrophobic Qo site by protons supplied from the oxidation of ubiquinol, independent of bulk pH. , contrary to , can be formed in non-aqueous environments (74, 75) and is readily membrane-permeable (14–17). The proton for the formation of would come from either a direct proton-coupled electron transfer (76) from neutral, singly protonated semiquinone or via shuttling of the proton from the acidic residues in the Qo site (77). Our experiments with varied pH led us to conclude that the former most likely occurs, which would also be consistent with the pH independence of the energy of activation of Complex III (78), indicating that during catalysis the Qo site does not equilibrate with bulk aqueous phase protons (27). Once formed in the Qo site, would then diffuse along the hydrophobic tunnel into the lipid phase, which opens up roughly in the middle of the inner mitochondrial membrane. From there, could diffuse to both sides of the inner mitochondrial membrane with more or less equal probability. We have previously discussed in detail the feasibility, in terms of thermodynamics and structural requirements, of the hypothesis (2).

Arguing every detail of this hypothesis is beyond the scope of the present work, but an immediate objection must be addressed to make the hypothesis credible. It could be argued that is too reactive to cross membranes since it would react with polyunsaturated fatty acids at diffusion-controlled rates. To counter this point, empirical evidence already attests that is readily able to cross membranes containing polyunsaturated fatty acids (14, 16), and the rate constant for reacting with linoleic, linolenic, and arachidonic acids is only ~103 M–1 s–1 (44, 79, 80), 6 orders of magnitude lower than the diffusion limit.

Future experiments will be needed to verify or falsify either of our proposed hypotheses. Nevertheless having demonstrated that the Qo site of Complex III releases superoxide toward both the mitochondrial matrix and the IMS, we argue that at present the two hypotheses we put forward best explain this observation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant PO1AG14674; a Veterans Affairs merit award (to H. V. R.); and National Institutes of Health Institutional training grant 5T3-AG021890-02 (to F. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Dr., MSC 7762, San Antonio, TX 78229-3900. Tel.: 210-885-6562; E-mail: mullerf{at}uthscsa.edu.

1 The abbreviations used are: SMP, submitochondrial particle; Q, ubiquinone; SOD, superoxide dismutase; HRP, horseradish peroxidase; cyt c, cytochrome c; MCLA, modified Cypridina luciferin analog; DHE, dihydroethidine; IMS, intermembrane space; Qo site, ubiquinol oxidation site; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide. Back


   ACKNOWLEDGMENTS
 
We thank Arlan Richardson for encouragement and support and Paul S. Brookes and Satomi Miwa for discussions and sharing unpublished observations.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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