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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;
* 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.
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.
The mitochondrial electron transport chain is the main source of ATP in the mammalian cell and thus is essential for life (
), forming the oxygen free radical superoxide (in its anionic form andin 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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,
)) 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.
). 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 (
) 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.
; 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 (
)), 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. (
), 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 (
)). 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 (
) 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 (
), confirming that Complex III-derived superoxide can indeed reach the matrix. We discuss two models of superoxide production that explain this observation.
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 (
). 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 (
). 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 × g, and the supernatant was passed through two cheesecloth layers and centrifuged at 14,000 × 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 (
). 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 Amplex™ red-horseradish peroxidase method (Molecular Probes, Eugene, OR (
). 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 allinto H2O2, a necessity sincereacts very rapidly with HRP and HRP-Compound I, resulting in underestimation of the actual rate of H2O2 production (
). 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) (
). 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 (
), 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-α]-pyrazin-3-one, Molecular Probes) is chemically very similar to coelenterazine (
). 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 (
). 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 (
). 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 ∼106m–1 s–1 (
), 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 (
). 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 (
), the results were expressed as a percentage relative to control, which, in our case, consisted of untreated skeletal muscle mitochondria.
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 (
). 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. (
) 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).
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 (
)). 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 (
) 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.
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.
). 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 (
). 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 (
). 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.
Vectorial Release of Complex I-derived Superoxide into the Matrix—It is generally accepted thatdoes not cross membranes. Empirical evidence backs up this view (
) 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 (
). 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 (
). 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 (
). 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.
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 (
), 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.
, 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.
Table IMN-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 extramitochondrialrelease 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 (
)), 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 (
)) 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 (
), 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 (
). 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 (
)). 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 (
), 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.
). 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 (
)), 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 extramitochondrialrelease, leaving ∼50% unaccounted for.
We hypothesized that the fraction of H2O2 production that could not be accounted for by extramitochondrialrelease 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 (
). 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 extramitochondrialwas 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 ofto be 4.9, only ∼1% of aqueous superoxide would be in theform at neutral pH (
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 information in the aqueous phase (i.e. IMS) cannot adequately explain this observation (we have discussed this in detail previously (
)). To explain that Qo site-derived superoxide reaches both sides of the inner mitochondrial membrane the model requires impermeableto somehow cross the inner mitochondrial membrane. This could not be accomplished through anion channels (sincewould have to move against the membrane potential) even if hypothetical anion channels capable of channeling superoxide did exist. Liu (
) suggested that aqueousin 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).
But we argue that two previously proposed hypotheses can accommodate these new data. Our first hypothesis (
) 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.
). 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 (
) thus suffering the same drawbacks. However, the critical difference between these models is that our hypothesis hasforming 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 (
). 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 thehypothesis (
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 thatis 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 thatis readily able to cross membranes containing polyunsaturated fatty acids (
), 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.
We thank Arlan Richardson for encouragement and support and Paul S. Brookes and Satomi Miwa for discussions and sharing unpublished observations.