Deleterious Role of Superoxide Dismutase in the Mitochondrial Intermembrane Space*

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1-/-) did not. Also, lymphocytes isolated from SOD1-/- mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.

strong nucleophile and weak reductant, with the ability to actively react with a number of cellular targets. Instead of reverse oxidation of superoxide to oxygen, the detoxifying mechanism for superoxide includes dismutation to hydroperoxide and oxygen. There are three dedicated enzymes catalyzing this dismutation reaction: copper-zinc superoxide dismutase (SOD1), which is localized mainly in the cytosol; manganese superoxide dismutase (SOD2), which is found in the mitochondrial matrix; and extracellular superoxide dismutase (SOD3) (3).
Unlike hydroperoxide, which freely diffuses through the membranes, superoxide cannot cross the mitochondrial inner membrane. In the matrix, SOD2 converts superoxide to hydroperoxide, which in turn is reduced to water by the matrix glutathione peroxidase (4). In the mitochondrial intermembrane space (IMS), superoxide is produced presumably by complex III (1). The fate of superoxide in this compartment is expected to be determined by SOD1 and cytochrome c, which is present in millimolar concentrations (5,6). Recently, cytosolic SOD1 was demonstrated in the yeast (7) and rat (8) IMS, where it has been suggested to be an important part of the mitochondrial superoxide-scavenging system by detoxification of ROS. Cytochrome c is a heme-containing protein that functions as an electron carrier between complex III and cytochrome oxidase in the respiratory chain. Cytochrome c can also efficiently oxidize superoxide to oxygen. In this respect, cytochrome c functions as a true antioxidant, scavenging superoxide without production of secondary ROS (9).
However, cytochrome c also has the potential to catalyze oxidation by hydroperoxide. Notably, hydroperoxide oxidizes the prosthetic heme in the cytochrome c molecule to oxoferryl heme, forming the so-called peroxidase compound I-type intermediate, a highly reactive oxidant that is able to react with a number of intracellular targets, including proteins, nucleic acids, and lipids, causing cell damage (10). Cytochrome c peroxidase activity is controlled by the coordination state of heme iron, particularly by the sulfur ligand of Met-80, which can be easily displaced by hydroperoxide (11,12). The peroxidase activity of cytochrome c is increased by unfolding and posttranslational modifications such as proteolytic cleavage, nitration, and oxidation (13)(14)(15).
We hypothesized that, upon mitochondrial stress, SOD1 might compete with cytochrome c for superoxide in the IMS and generate hydroperoxide, which then could react with cytochrome c and form the peroxidase compound I-type interme-* 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.

EXPERIMENTAL PROCEDURES
Animals-The experiments were conducted according to the Council of Europe (Directive 86/609) and Finnish guidelines and approved by the State Provincial Office of Eastern Finland. 8-week-old male C57BL mice were decapitated under carbon dioxide anesthesia, and mitochondria from liver tissues were isolated. SOD1 knock-out (SOD1 Ϫ/Ϫ ) mice with a 129/CD1 background were bred with the CD1 strain of mice for at least eight generations. G93A SOD1 transgenic mice (B6.Cg-Tg-(SOD1-G93A)1Gur/J, The Jackson Laboratory, Bar Harbor, ME) carrying a high copy number of the human mutant G93A SOD1 were killed at 8 weeks of age.
Isolation of Mitochondria-The liver tissue was homogenized using an all-glass Dounce homogenizer (Kontes) in icecold isolation buffer (320 mM sucrose, 1 mM EGTA, and 10 mM Tris-HCl, pH 7.4). The homogenate was centrifuged for 3 min at 2000 ϫ g, and the supernatant was transferred to a new tube and centrifuged for 10 min at 10,000 ϫ g. The resulting supernatant was discarded, and the pellet-containing mitochondrial fraction was washed once with wash buffer (0.2 M sucrose, 20 mM HEPES, pH 7.2, 0.1 mM EGTA, and 4 mM KH 2 PO 4 ) and resuspended in the same buffer to a protein concentration of 10 mg/ml. The protein concentrations were determined using Bio-Rad protein assay dye. For the ROS production studies, mitochondria were resuspended to a protein concentration of 10 mg/ml in standard medium (0.3 M mannitol, 10 mM KCl, 10 mM KH 2 PO 4 , 5 mM MgCl 2 , and 1 mg/ml bovine serum albumin, pH 7.4). The purity of the mitochondrial fraction obtained was assessed by Western blotting (supplemental Fig. 1). The intactness and functional integrity of the isolated mitochondria were confirmed by measuring mitochondrial membrane potential with JC-1 dye (5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolylcarbocyanine iodide) and mitochondrial respiration with an oxygraph method (supplemental Fig. 2).
Isolation of Mitoplasts-To obtain mitoplasts, mitochondria were incubated with 5ϫ volume of cold hypotonic buffer (10 mM Tris, pH 7.4, 1 mM EDTA, and 1 mM dithiothreitol) for 10 min on ice. After 10 min, 150 mM NaCl was added, and mitoplasts were incubated for 10 min on ice and centrifuged at 18,000 ϫ g for 20 min at 4°C. Mitoplasts were washed and resuspended back to the original volume in standard medium.
Measurements of Mitochondrial ROS Production-Measurements were carried out in 96-well plates by mixing 20 l of mitochondrial suspension (containing 150 g of mitochondrial protein) with 140 l of standard medium and adding substrate mixture to final concentrations of 1.3 mM succinate and 10 M 2,7-dichlorodihydrofluorescein diacetate (DCF) (Fluka). Antimycin A (at a 3 M final concentration; Sigma) was added in experiments in which respiratory chain complex III was inhibited. Oxidized DCF fluorescence was recorded with a VICTOR multilabel reader (Wallac). The DCF oxidation rate in the absence of mitochondria was also measured, and even though the reagents (especially SOD1 and cytochrome c) increased the DCF oxidation rate in the absence of mitochondria, the rate was only up to 10% of the rate measured in the presence of mitochondria (supplemental Fig. 3). All samples were run in triplicates.
Measurements of Cytochrome c-catalyzed Peroxidation-To study the role of SOD1 in cytochrome c-catalyzed DCF oxidation in the presence of a superoxide source, 15 l of xanthine oxidase suspension (diluted 1:100 in 50 mM phosphate buffer, pH 7.8; Sigma) was mixed with xanthine solution to a 0.4 mM final concentration and complemented with 10 M DCF. The total volume of the reaction mixture was 200 l. DCF oxidation was recorded using the VICTOR multilabel reader. The fluorescence kinetics was recorded in the presence of 10 M cytochrome c (Sigma) and together with purified human erythrocyte SOD1 (15,30,60, and 250 nM). Human SOD1 was isolated from erythrocytes by fractionated precipitation and DEAE ion exchange chromatography (16). The purity and identity of isolated SOD1 were confirmed by SDS-PAGE and immunoblotting. All samples were run in triplicates.
IMS Isolation and Measurement of SOD1 Activity-SOD1 activity was measured in the mitochondrial IMS preparation (obtained in the presence of iodoacetamide) by nitro blue tetrazolium reduction assay and was expressed as a percentage of activated enzyme in the absence of iodoacetamide. To isolate the contents of the IMS, mitochondria (10 mg/ml) were treated with 0.1 mg of digitonin/mg of mitochondria for 1 h at room temperature. 100 mM iodoacetamide was added to samples to prevent SOD1 activation upon disruption of the outer membrane. After centrifugation at 10,000 ϫ g for 10 min, the supernatant was assayed for SOD activity as quenching of nitro blue tetrazolium reduction by xanthine oxidase/xanthine reactiongenerated superoxide anion radical (17,18). SOD1 activity was measured in the mitochondrial IMS preparation (obtained in . ) is released in the IMS by one-electron reduction of oxygen at a site in the inner membrane (step I). Cytochrome c (CytC) oxidizes superoxide to oxygen (step II). SOD1 in the IMS is activated by oxidation of sulfhydryl groups, leading to formation of intramolecular SϭS bonds (step III). SOD1 produces hydroperoxide (H 2 O 2 ) by dismutating superoxide. Hydroperoxide oxidizes cytochrome c to oxoferryl cytochrome c (CytC(Fe 4ϩ ϭO)), an exceptionally strong oxidant able to oxidize a number of vital biological targets (step V).
the presence of iodoacetamide) and was expressed as a percentage of activated enzyme in the absence of iodoacetamide (8).
Kinetics of DCF Oxidation by Isolated Mitoplasts-ROS production by mitoplasts was measured in the presence of 1.3 mM succinate, 10 M DCF, 3 M antimycin A, and 10 M cytochrome c as described above for ROS production by mitochondria. 100 nM purified human erythrocyte SOD1 was added to study the effect of SOD1 on ROS production.
Flow Cytometry-Peroxide production in mouse blood lymphocytes in the presence of 3 M antimycin A loaded with 10 M DCF was analyzed by flow cytometry (FL1 Ϫ DCF fluorescence). Mouse (wild-type and SOD1 Ϫ/Ϫ ) blood lymphocytes were isolated by differential centrifugation using Ficoll-Paque PLUS (GE Healthcare) according to the manufacturer's instructions. Lymphocytes were washed and suspended in Hanks' balanced salt solution containing 10 M DCF. 10 min later, 3 M antimycin A was added, and the samples were immediately analyzed using a FACSCalibur flow cytometer (BD Biosciences) at 30-and 90-min time points. 10,000 cells were counted altogether.
The kinetics of antimycin A-induced apoptosis in lymphocytes was measured by annexin V-fluorescein isothiocyanate binding. Mouse (wild-type and SOD1 Ϫ/Ϫ ) blood lymphocytes were isolated by differential centrifugation using Ficoll-Paque PLUS according to the manufacturer's instructions. Lymphocytes were washed and suspended in Hanks' balanced salt solution containing 20 M antimycin A. Untreated cells and antimycin A-treated lymphocytes after 30, 60, and 90 min were sedimented by centrifugation and resuspended in binding buffer containing 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl 2 , pH 7.4. Annexin V-fluorescein isothiocyanate (Sigma) was added to a final concentration of 0.45 g/ml, and after incubation for 10 min, propidium iodide was added to a final concentration of 2 g/ml. After another 10-min incubation, the samples were analyzed using the FACSCalibur flow cytometer. 10,000 cells were counted altogether.
Zymography-SOD activity in the IMS preparations was assessed as described (18). Briefly, 10 l of each sample (containing 0.2 g/l total protein) was loaded onto a 10% native polyacrylamide gel. After electrophoresis, the gels were washed with 50 mM phosphate buffer, pH 7.8, for 10 min and then incubated in 1 mg/ml nitro blue tetrazolium solution in the same buffer for 15 min. After incubation, the gels were briefly washed with phosphate buffer and incubated in 0.25% TEMED solution containing 30 M riboflavin for 15 min. The gels were rinsed in phosphate buffer and illuminated for 15 min with a fluorescent light source. SOD activity appeared as clear bands on a blue background. The gels were scanned with a Gel Doc scanner (Bio-Rad), and the bands were quantified with Image-Quant software (GE Healthcare). In case of mitoplast analysis, equal loading was assured by equal SOD2 activity bands. Samples were run in triplicates.
Statistical Analyses-Results are shown as the means Ϯ S.D. Differences between groups were determined by Student's t test. p Ͻ 0.05 was considered statistically significant.

SOD1 Controls Mitochondrial Hydroperoxide Production, and Its Substrate Oxidation Is Dependent on Cytochrome c-
The isolated mouse liver mitochondria contained a considerable amount of SOD1. When analyzing the distribution of SOD1 in different subcellular fractions, the SOD1 concentration in the mitochondria was ϳ30% (per unit of total protein) of the cytosolic SOD1 concentration. Within mitochondria, the SOD1 concentration was about six times higher in the IMS fraction than in the mitoplast fraction (supplemental Fig. 4). To investigate whether SOD activity in the IMS contributes to hydroperoxide production, mitochondria respiring in the presence of succinate were challenged with antimycin A, an inhibitor of complex III. Antimycin A has been shown previously to cause prompt superoxide production as determined by EPR (19). SOD activity in an IMS preparation was rapidly increased as a function of time in response to antimycin A ( Fig. 2A). Inhibition of mitochondrial respiration also resulted in increased hydroperoxide production as determined in parallel by measurement of the fluorescence of DCF, a widely used hydroperoxide-sensitive probe (20). DCF is strongly oxidized to a fluorescent derivative in the presence of heme-containing catalysts such as cytochrome c (10). In parallel, the hydroperoxide produced by mitochondria was measured using a luminol/horseradish peroxidase assay and found to linearly correlate with the amount of mitochondrial proteins (supplemental Fig. 5). The possibility that DCF could also be oxidized by peroxynitrite after reaction of superoxide with nitric oxide was ruled out because inhibition of nitric-oxide synthase by N -nitro-L-arginine did not alter the DCF fluorescence rate (supplemental Fig.  6). The monitored fluorescence is thus a measure of the relative oxidative damage to cell constituents. An increase in DCF fluorescence was observed after a lag period in respiring isolated mitochondria. The lag period was remarkably shortened by antimycin A (Fig. 2C), and the response coincided with the maximal increase in SOD activity. In agreement with a previous study (8), these kinetics of hydroperoxide production and SOD1 activity suggest a redox control of the dismutase activity in the IMS. SOD1 was essential for the increased hydroperoxide production because adding the SOD1 inhibitor ammonium tetrathiomolybdate or ammonium diethyldithiocarbamate significantly and dose-dependently reduced DCF fluorescence in isolated mitochondria (Fig. 2B and supplemental Fig. 7).
These results led us to the paradoxical hypothesis that mitochondria lacking SOD1 should produce less hydroperoxide when stressed by inhibition of respiration, resulting in lower cytochrome c-catalyzed DCF oxidation. To test this hypothesis, we isolated intact mitochondria from wild-type and SOD1 Ϫ/Ϫ mice. Mitochondrial peroxide production was measured again by DCF oxidation in the presence and absence of antimycin A. Indeed, the mitochondria isolated from SOD1-deficient (SOD1 Ϫ/Ϫ ) mice produced substantially less DCF fluorescence and did not show any response to inhibition of complex III by antimycin A (Fig. 2C).
These results indicate that, upon inhibition of mitochondrial respiration, elevated SOD1 activity is responsible for increased hydroperoxide production in the IMS, resulting in cytochrome c-catalyzed DCF oxidation. To test this hypothesis further, we used mitoplasts, e.g. mitochondria devoid of outer membrane and IMS, to reconstitute conditions for hydroperoxide production. No significant DCF oxidation could be detected in respiring mitoplasts even after inhibition of complex III by antimycin A (Fig. 2D). Addition of cytochrome c caused an increase in DCF fluorescence, possibly because of the reconstituted respiration and hydroperoxide escaping from the mitochondrial matrix. Notably, addition of 100 nM SOD1 more than doubled the rate of DCF oxidation in the presence of cytochrome c (Fig. 2D).
To model the interaction of superoxide, cytochrome c, and SOD1 in the IMS, we reconstituted a reaction in which superoxide was generated through xanthine oxidase/xanthine. A slow oxidation of DCF occurred in the presence of this enzyme substrate pair. The rate of DCF oxidation was slightly elevated by 5 M cytochrome c (Fig. 2E). However, addition of increasing concentrations of SOD1 to the reaction mixture strongly and dose-dependently increased the rate of DCF oxidation, indicating that SOD1 significantly enhances cytochrome c-catalyzed peroxidation. The effect of SOD1 was hydroperoxide-dependent because addition of 5 units/ml catalase completely abolished it (supplemental Fig. 8).
SOD1 Increases Mitochondrial Peroxide Production in Lymphocytes and Contributes to Apoptosis-To investigate whether SOD1 also controls hydroperoxide production in the IMS of intact cells, we isolated lymphocytes from wild-type and SOD1 Ϫ/Ϫ mouse blood by differential centrifugation and loaded them with DCF before adding antimycin A. As shown in Fig. 3 (A and B), flow cytometric analysis showed that antimycin A-induced DCF oxidation was attenuated in SOD1 Ϫ/Ϫ lymphocytes at 90 min of incubation compared with hydroperoxide production in wild-type lymphocytes. The difference in hydroperoxide production was confirmed to be statistically significant by the Kolmogorov-Smirnov test.
In addition to increased superoxide production in mitochondria, inhibition of complex III by antimycin A has also been shown to induce apoptosis (21). To test whether SOD1 activity is important for apoptosis, SOD1 Ϫ/Ϫ and wild-type lymphocytes were challenged with antimycin A for 90 min, and apoptosis was determined by annexin V binding using flow cytometry. SOD1 Ϫ/Ϫ lymphocytes showed significantly less apoptosis than did wild-type lymphocytes (Fig.  3 (C and D) and supplemental Fig. 9).
Increased SOD1 Activity Associated with G93A SOD1 in Spinal Cord Mitoplasts Leads to Elevated ROS Production-Mutations in SOD1 are known to cause the familial form of amyotrophic lateral sclerosis (ALS) (22), a disease characterized by loss of motor neurons in the spinal cord, leading to muscle atrophy, paralysis of voluntary muscles, and death in 3-5 years (23). Although the nature of the toxic gain of function in mutant SOD1 has not been identified, it is believed that altered generation of free radical and ROS is a leading contributor to the destruction of motor neurons. The most recent studies of familial ALS animal models indicate that mutant SOD1 causes selective mitochondrial dysfunction, resulting in increased ROS production. We hypothesized that the increased amount of SOD1 may be associated with SOD1 activity in the mitochondrial IMS and thereby boost ROS production.

FIGURE 2. SOD1 in the IMS controls cytochrome c-catalyzed peroxidation.
A, the burst of hydroperoxide production in wild-type mitochondria (f) coincides with the peak of SOD1 activity in the IMS (OE). SOD1 activity was measured in the mitochondrial IMS preparation (obtained in the presence of iodoacetamide) by nitro blue tetrazolium reduction assay and is expressed as a percentage of fully activated enzyme in the absence of iodoacetamide. RU, response unit. B, inhibition of SOD1 activity reduces mitochondrial hydroperoxide production. Mitochondrial hydroperoxide production by mitochondria isolated from the livers of C57BL male mice was measured in the presence of 0, 10, and 50 M tetrathiomolybdate (TTM). C, mitochondrial hydroperoxide production measured as DCF oxidation in wild-type and SOD1 Ϫ/Ϫ mitochondria. In wild-type mitochondria (wt; F), hydroperoxide production was accelerated by antimycin A (AA; f). In contrast, SOD1 Ϫ/Ϫ mitochondria (knock-out (ko); ) did not respond to antimycin A (OE) and produced significantly less hydroperoxide. D, rate of DCF oxidation by isolated mouse liver mitoplasts in the presence of 10 M cytochrome c and upon addition of 100 nM SOD1. Addition of SOD1 caused a significant increase in the hydroperoxide production rate (p ϭ 0.007). In the absence of cytochrome c, SOD1 only slightly increased the DCF oxidation rate. E, SOD1 controls cytochrome c-catalyzed peroxidation in vitro in a concentration-dependent manner. Slow DCF oxidation by superoxide (generated by xanthine oxidase (XO)/xanthine; Š) was slightly enhanced by 5 M cytochrome c ((CytC); ࡗ). SOD1 at 15 nM (), 30 nM (OE), 60 nM (F), and 250 nM (f) significantly increased the rate of DCF oxidation. Cntr, control.
Thus, we studied mitochondrial SOD1 activity in transgenic mice expressing mutant G93A SOD1, a model of familial ALS (24). The SOD1 mutant accumulates in mitochondria of the spinal cord, where it is associated with the inner membrane (25)(26)(27). The spinal cord is also the most affected tissue in ALS. The SOD1 activity of IMS preparations from the spinal cord was 6-fold greater in the G93A SOD1 mice than in the wild-type mice (Fig. 4, A and D). In addition, IMS SOD1 activity in the spinal cord was clearly higher in comparison with non-affected brain tissues of the same animals (Fig. 4, B and E). The SOD1 activity of the mitoplast preparations isolated from the transgenic spinal cord was twice as high as that from the transgenic brain (Fig. 4, C and F). To investigate whether the elevated activity and accumulation of mutant G93A SOD1 in mitoplasts result in increased ROS production, we measured DCF oxidation in the mitoplast preparations in the presence of cytochrome c. We found that the mitoplasts isolated from the transgenic spinal cord produced significantly more DCF fluorescence compared with the mitoplasts derived from the transgenic cortex (Fig. 4G), indicating that the increased amount of SOD1 in the IMS is also associated with an increase in hydroperoxide production.

DISCUSSION
Our data indicate that inhibition of electron transfer at the level of complex III leads to an increase in SOD1 activity in the IMS, paradoxically resulting in increased hydroperoxide production and consequently cytochrome c-catalyzed peroxida-tion. This could trigger a vicious circle where oxidative damage to mitochondrial respiratory components leads to further ROS production and peroxidation. Indeed, we have demonstrated that inhibition of mitochondrial respiration at the level of complex III causes SOD1-dependent ROS production and apoptotic death of isolated blood lymphocytes. Moreover, accumulation of human mutant G93A SOD1 in the IMS that is observed in the transgenic animal models of ALS leads to elevated SOD1 activity and increased cytochrome c-catalyzed oxidation in the IMS.
SOD is generally thought to protect cells from oxidative damage. Accordingly, as a cytosolic antioxidant, SOD1 provides protection in models of transient myocardial (28) and brain (29) ischemia and Parkinson disease (30). Some other studies suggest, however, that the increased SOD1 activity promotes injury. For instance, immature mouse brains overexpressing SOD1 show an increased propensity for injury and accumulate more hydroperoxide after hypoxia-ischemia than do mouse wild-type brains (31). Also, elevation of SOD1 increases acoustic trauma from noise exposure (32), and mice deficient in SOD1 are resistant to acetaminophen toxicity (33).
Even though SOD1 as a cytosolic antioxidant protects against mitochondrial dysfunction in a mouse model of transient focal cerebral ischemia (34), SOD1 deficiency (rather than overexpression) is associated with enhanced recovery and attenuated activation of NF-B after brain trauma in mice (35). Moreover, a superoxide generator, menadione, produces sig-  . Increased SOD1 activity in the mitochondria of mouse mutant G93A SOD1 spinal cord leads to elevated ROS production. SOD1 activity was measured by in-gel zymography. A and D, SOD1 activity in the IMS of mitochondria isolated from the spinal cords (Sc) of mutant G93A SOD1 mice increased by nearly 600% in comparison with the wild-type (wt) animals. *, p ϭ 0.02. tg, transgenic. B and E, SOD1 activity in the mitochondrial IMS from the spinal cords of mutant G93A SOD1 mice increased in comparison with the SOD1 activity in the IMS from the brain (Br) tissue of the same animals. **, p ϭ 0.0068. C and F, SOD1 activity associated with isolated mitoplasts from the spinal cords of mutant G93A SOD1 mice was significantly increased compared with that associated with mitoplasts from the brains of the same animals. *, p ϭ 0.017. G, isolated mitoplasts from the spinal cords of mutant G93A SOD1 mice produced significantly more ROS in the presence of cytochrome c compared with the mitoplasts from the brains of the same animals. **, p ϭ 0.037. RU, response unit. nificantly increased DCF fluorescence and greater death in SOD1 transgenic neurons than in wild-type neurons, suggesting increased hydroperoxide formation in the SOD1 transgenic cells (36). This apparent discrepancy concerning the role of SOD1 in cell injury can be explained by the results presented here showing that increased SOD1 activity in the IMS paradoxically produces peroxides that are converted to highly toxic ROS.
Previous studies, including EPR studies (12,(37)(38)(39), have demonstrated that the reaction of cytochrome c with hydroperoxide results in formation of oxoferryl cytochrome c (peroxidase compound I-type intermediate) and the corresponding protein-derived tyrosyl radical, which is highly reactive and has the potential to oxidize proteins, DNA, and lipids as well as endogenous antioxidants such as glutathione, NADH, and ascorbate (10). In particular, oxidation of cardiolipin (a phospholipid that is in complex with cytochrome c on the surface of the inner mitochondrial membrane) causes the release of proapoptotic factors from mitochondria (37,40). This leads to a scenario in which the hydroperoxide produced by increased SOD1 activity in the IMS would thus serve as a substrate for cardiolipin-bound cytochrome c and consequently switch on very early pro-apoptotic processes, inducing consecutive programmed cell death (38).
On the other hand, oxidized cytochrome c (Fe 3ϩ ) can efficiently scavenge superoxide (supplemental Fig. 10). At a high physiological concentration of cytochrome c in the IMS, the superoxide released into this compartment might become oxidized to oxygen without production of secondary ROS (hydroperoxide). Also, export of superoxide via voltagedependent anion channels (41) would serve as a safe pathway for the superoxide produced in the IMS. In contrast, competitive superoxide dismutation catalyzed by SOD1 or occurring non-enzymatically (42) leads to hydroperoxide production in the IMS, which most likely is harmful to the mitochondria and the cell. Altogether, the data indicate that hydroperoxide in the presence of peroxidase activity may play a key role in oxidation of biological targets in the IMS. Thus, SOD1 activity (supplemental Fig. 11) and other factors that may lead to increased hydroperoxide production in this compartment can be regarded as deleterious to the mitochondria and the cell.
Mitochondrial dysfunction (including altered function of respiratory complexes) has been described in arteriosclerosis, diabetes mellitus, and a number of acute and degenerative brain diseases such as stroke, Parkinson disease, and ALS. Increased ROS production is also a characteristic of these diseases (43). Increased SOD1 activity accompanied by high hydroperoxide production in the IMS may be one mechanism of neurodegeneration. Notably, the toxicity of ALS-linked SOD1 mutants originates from their selective recruitment to spinal cord mitochondria (25). Even though we clearly showed that mutant G93A SOD1 is able to associate with the mitochondrial inner membrane specifically in the spinal cord, the molecular reason for this effect remains unknown, but may be dependent on oxidation of Cys residues in mutant SOD1 (44). In conclusion, we suggest that SOD1 activity in the IMS may be a relevant therapeutic target for many degenerative diseases involving mitochondrial pathogenesis.