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J. Biol. Chem., Vol. 279, Issue 6, 4127-4135, February 6, 2004

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Characterization of Superoxide-producing Sites in Isolated Brain Mitochondria^*

Alexei P. Kudin, Nana Yaw-B. Bimpong-Buta¶, Stefan Vielhaber, Christian E. Elger¶, and Wolfram S. Kunz¶||

From the ^¶Department of Epileptology, University Bonn Medical Center, Sigmund-Freud-Str. 25, D-53105 Bonn and the Department of Neurology, University Magdeburg Medical Center, Leipziger Str. 44, D-39120 Magdeburg, Germany

Received for publication, September 17, 2003 , and in revised form, October 27, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD⁺/NADH redox couple on H₂O₂ and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H₂O₂ (0.04 ± 0.02 nmol H₂O₂/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 ± 0.25 nmol H₂O₂/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 ± 0.27 nmol H₂O₂/min/mg). The maximal rate of H₂O₂ generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 ± 0.07 nmol H₂O₂/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to –295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H₂O₂ induced increased H₂O₂ production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Superoxide anion, a product of one-electron reduction of oxygen, is the by-product of normal functioning of the mitochondrial respiratory chain (1). It has been reported, that this radical is generated by complexes I and III of the mitochondrial respiratory chain and readily converted to H₂O₂ by mitochondrial Mn-superoxide dismutase (2–6). There is substantial evidence that superoxide and H₂O₂ contribute to the pathogenesis of certain neurodegenerative diseases (7, 8). However, there is considerable disagreement in recent literature concerning generated amounts and sites of superoxide production by isolated mitochondria. In contrast to the well documented production of superoxide at center "o" of antimycin A-inhibited complex III (2, 9, 10), the exact site and the total contribution of reactive oxygen species (ROS)¹ generation in complex I has not been established so far. Although certain investigators (11, 12) suggested low potential iron-sulfur clusters as potential sites, others (6) proposed flavine mononucleotide (FMN) to be the producer of superoxide being responsible for the H₂O₂ generation by brain mitochondria. In addition, there are substantial controversies regarding the exact amounts of ROS production at the different sites of respiratory chain. Although some reports document the highest rates of H₂O₂ production at a site at respiratory chain complex I (5, 12) others point to complex III as the main contributor (6, 10). Therefore, we extensively characterized ROS production at the different generation sites in respect to oxygen consumption of well coupled isolated rat and human brain mitochondria. Our results indicate that most of the superoxide radicals are produced at complex I and that a high production of reactive oxygen species is a feature of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. Because the redox potential of the superoxide production at complex I was determined to be –295 mV, this suggests that the generation site is most likely the FMN moiety. Moreover, short-term incubation of rat brain mitochondria with H₂O₂ in the lower mM range was observed to induce increased H₂O₂ production at this site, creating a self-accelerating vicious cycle. This mechanism is proposed to be important in brain pathology associated with mitochondrial damage and neuronal cell death.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Isocitrate dehydrogenase was purchased from Boehringer (Mannhein, Germany); L-adrenaline and bacterial protease (nagarse) from Fluka (Buchs, Switzerland); superoxide dismutase and digitonin from Serva (Heidelberg, Germany); and monochlorobimane from Molecular Probes (Leiden, The Netherlands). All other chemicals were obtained from Sigma-Aldrich.

Solutions—Solutions include the following: MSE solution (225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM HEPES, 1 mg/ml BSA, pH 7.4); MSE-nagarse solution (0.05% nagarse in MSE solution); MSE-digitonin solution (0.02% digitonin in MSE solution); and MTP medium (10 mM KH₂PO₄,60mM KCl, 60 mM Tris-HCl, 110 mM mannitol, 0.5 mM EDTA, pH 7.4).

Isolation of Rat Brain Mitochondria—Mitochondria from one rat brain were isolated according to the protocol described by Rosental et al. (13) with small modification that allowed to obtain mitochondria with much better functional characteristics. In brief, the isolation protocol was the following. Before mitochondrial isolation all solutions were cooled down until the slight appearance of ice. One Wistar rat (60–90 days old) was anesthetized by chloroform and killed by decapitation. Its brain was immediately transferred into the ice-cold MSE solution and shaken to wash out blood. Then we minced the brain, added 10 ml of ice-cold MSE-nagarse solution, and homogenized it at 600 units/s using a potter homogenizer. Thereafter, we added 20 ml of ice-cold MSE solution and centrifuged the homogenate at 2000 x g for 4 min. After centrifugation we passed the supernatant through a cheesecloth and centrifuged it at 12,000 x g for 9 min. To permeabilize synaptosomes we dissolved the resulting pellet with 10 ml of ice-cold MSE-digitonin solution, transferred the solution to a small glass homogenizator, and homogenized it 8–10 times manually to obtain a homogenous suspension. Finally, we centrifuged the suspension at 12,000 x g for 11 min and dissolved the resulting pellet in about 300 µl of MSE solution to obtain about 20 mg protein/ml.

Isolation of Human Brain Mitochondria—Tissue samples from human parahippocampal gyrus were obtained from 9 patients (7 female and 2 male) with therapy-resistant temporal lobe epilepsy, who underwent epileptic surgery. White matter was rapidly removed and the remaining gray matter sample (about 150–200 mg wet weight) was immediately transferred into ice-cold MSE solution. The remaining isolation procedure of mitochondria was identical to the protocol described above. All patients gave written informed consent, and the study was approved by the University of Bonn Ethical Committee.

Preparation of Submitochondrial Particles—Frozen rat brain mitochondria (protein concentration of about 20 mg/ml) were thawed and sonicated three times for 15 s by the ultrasonic processor GEX-600. The further preparation was performed as described in Ref. 14.

Respiration—The oxygen consumption of mitochondria was determined at 30 °C in MTP medium with a PC-supported Oroboros high resolution oxygraph (15).

Measurement of H₂O₂ Production—Mitochondrial H₂O₂ generation was measured by monitoring the change in fluorescence of 200 µM p-hydroxyphenylacetic acid (_ex = 317 nm, _em = 414 nm) catalyzed by 20 units/ml horseradish peroxidase (6). Different O₂ concentrations were achieved by mixing argonand oxygen-saturated buffers. Oxygen concentrations were calculated from the O₂ solubility in air and oxygen-saturated MSE solution at 30 °C and 101 kPa (0.23 and 1.15 mM, respectively), and from mixing ratios of the argon and oxygen-saturated solutions.

Measurement of Superoxide Production—The -dependent oxidation of 1 mM epinephrine to adrenochrome in the presence of 7800 units/ml catalase was followed spectrophotometrically at 486–575 nm with a dual wavelength spectrophotometer (Aminco DW 2000, SLM Instruments). In control experiments we monitored the change in fluorescence of p-hydroxyphenylacetic acid (_ex = 317 nm, _em = 414 nm) catalyzed by horseradish peroxidase (conditions of measurement as described above) in the additional presence of 22 units/ml Cu,Zn-superoxide dismutase.

Determination of Enzymatic Activities—Aconitase activity was measured according to Ref. 16 with small modifications. The mitochondrial suspension was diluted 10 times in a solution, containing 50 mM Tris-Cl and 0.6 mM MnCl₂, pH 7.4, just before the activity measurement. Then mitochondria were sonicated for 15 s with the ultrasonic processor GEX-600. Aconitase activity was determined at 30 °C using a dual-wavelength spectrophotometer (Aminco DW 2000, SLM Instruments) following the absorbance change at 340–380 nm (_red-ox = 5.5 mM^–1 cm^–1). The reaction mixture contained 50 mM Tris-Cl, pH 7.4, 5 mM sodium citrate, 0.6 mM MnCl₂, 0.2 mM NADP⁺, 0.1 mg/ml isocitrate dehydrogenase, 0.2% laurylmaltoside, and 40–70 µg/ml of protein. The activity of citrate synthase was determined by a standard method and the activity of NADH:coenzyme Q₁-reductase was measured at 340 nm-380 nm (_red-ox = 5.5 mM^–1 cm^–1); in a buffer containing 50 mM KCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 7.4, in the presence of 150 µM NADH, 100 µM coenzyme Q₁, and 2 mM KCN with a dual-wavelength spectrophotometer (Aminco DW 2000, SLM Instruments) (17). The protein content of mitochondria was determined using a protein assay kit based on Peterson's modification of the micro-Lowry method according to the instructions of the manufacturer (Sigma).

Determination of GSH Content—GSH content was determined using the method described in Ref. 18 with small modifications. The mitochondrial suspension was dissolved in MTP medium at a final concentration of 0.8 mg/ml. After addition of monochlorobimane (100 µM) and glutathione S-transferase (1 units/ml) the reaction mixture was sonicated for 15 s with the ultrasonic processor GEX-600. After 30 min development of reaction the mitochondrial suspension was centrifuged at 16,000 x g for 7 min. Thereafter, the fluorescence of each supernatant was measured (_ex = 380 nm, _em = 470 nm) using a fluorescence reader (Spectra MAX Gemini, Molecular Devices). As standard we used GSH dissolved in MTP medium in a range of 0 to 100 µM.

Statistics—All data are presented as means ± S.D. and p values smaller than 0.05 (according to two-sided t test) were considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Oxygen Dependence of H₂O₂ Generation by Mitochondria Isolated from Brain Tissue—Fig. 1 shows the measurement of generation of hydrogen peroxide by isolated rat brain mitochondria using the fluorescent probe p-hydroxyphenylacetate. Addition of mitochondria (BM) led to transient increase in fluorescence, which returned to a stable stationary level. The addition of the respiratory substrates glutamate+malate resulted in a low rate of fluorescence increase indicating the generation of only small amounts of hydrogen peroxide (GLU+MAL, upper trace; 1.6 pmol H₂O₂/min or 0.02 nmol H₂O₂/min/mg protein). An about 20-fold higher slope of the fluorescence trace was observed after the addition of the complex I inhibitor rotenone (ROT, upper trace; 37 pmol H₂O₂/min or 0.53 nmol H₂O₂/min/mg protein) or in the presence of the respiratory substrate succinate alone (SUCC, lower trace; 46 pmol H₂O₂/min or 0.66 nmol H₂O₂/min/mg protein). The addition of the inhibitor of bc₁ complex antimycin A in the presence of succinate led to an inhibition of H₂O₂ production (ANTI-A, lower trace; 7.6 pmol H₂O₂/min or 0.11 nmol H₂O₂/min/mg protein). This is an indication that in rat brain mitochondria most of the superoxide radicals are produced at complex I and that a high production of reactive oxygen species is a feature of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. Because it is well documented that mitochondrial superoxide production is strongly dependent on oxygen concentration (19, 20) we determined the oxygen concentration dependence of H₂O₂ production of rat brain mitochondria in the presence of the respiratory substrates glutamate+malate (Fig. 2A, filled circles), glutamate+ malate+rotenone (Fig. 2A, open circles), and succinate (Fig. 2B, filled circles). We detected hyperbolic concentration dependences from which we calculated a K_m value for oxygen of 0.92 ± 0.01 mM. A similar dependence was also observed for the production of H₂O₂ by complex III determined in the presence of succinate+antimycin A (data not shown).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Experimental traces of H2O2 generation by isolated rat brain mitochondria in the presence of different mitochondrial substrates. H2O2 generation was measured fluorimetrically determining the oxidation of p-hydroxyphenylacetic acid (200 µM) in oxygen saturated MTP medium in the presence of horseradish peroxidase (20 units/ml). Vertical arrow, fluorescence change caused by addition of 0.1 nmol H2O2 to the mitochondrial suspension. For A, BM, rat brain mitochondria (0.07 mg protein/ml); GLU+MAL, glutamate (10 mM) and malate (5 mM); ROT, rotenone (6.7 µM). For B, SUCC, succinate (10 mM); ANTI-A, antimycin A (0.5 µM).

View larger version (13K): [in this window] [in a new window]   FIG. 2. O2 dependence of H2O2 generation by rat brain mitochondria. A and B, about 0.2 mg protein/ml in the presence of glutamate (10 mM) and malate (5 mM) (A, filled circles, dotted curve) and plus rotenone (6.7 µM) (A, open circles, solid curve) and in the presence of succinate alone (10 mM) (B). Different values of O2 saturation were obtained by mixing of MTP medium gassed with pure oxygen (100% saturation) and gassed with argon (0% saturation). The individual data points were obtained in experiments with four independent mitochondrial preparations. C, original experimental traces of detection of complex I activity using the NADH absorbance measurement at 340–380 nm. Trace 1, MTP medium gassed with air (20% O2); traces 2 and 3, MTP medium gassed with 100% O2. Additions: BM, brain mitochondria (0.7 mg protein/ml); ROT, rotenone (20 µM); DPI, 10 µM diphenyleneiodonium; CMB, 0.5 µM p-chloromercuriobenzoate. The depicted numerical values represent the slope of each trace (in A/min).

The Maximal Rates of H₂O₂ Generation of Mitochondria Isolated from Human and Rat Brain Tissue Depend on Specific Electron Transport Activity—A possible reason for the discrepant results reported about ROS production of isolated brain mitochondria (5, 6, 12) could be variable mitochondria quality. Therefore, we checked the isolated mitochondria in the present investigation with regard to basic characteristics: respiration rates in different functional states and respiratory control values. In Fig. 3, typical oxygraph traces of mitochondria isolated from rat brain (A) and human parahippocampal cortex (B) are given. Using glutamate and malate as respiratory substrates, active state respiration rates of 231 ± 40 nmol O₂/min/mg protein (n = 21) at an average respiratory control ratio (RCR) = 5.4 ± 0.8 were obtained for rat brain mitochondria and 88 ± 25 nmol O₂/min/mg protein (n = 9) at RCR = 3.2 ± 0.6 for mitochondria of human parahippocampal cortex, respectively. With succinate (+rotenone) as substrate we obtained active state respiration rates of 210 ± 25 nmol O₂/min/mg protein (n = 13) for rat brain mitochondria and 76 ± 22 nmol O₂/min/mg protein (n = 8) for mitochondria of human parahippocampal cortex. These values indicate that both brain mitochondria preparations are tightly coupled, but human parahippocampal mitochondria appear to be less pure in respect to respiratory chain activity. Comparing the maximal H₂O₂ production rates (cf. Table I and Fig. 4A) rat brain mitochondria (Fig. 4A, white bars) seem to produce much higher quantities of H₂O₂ in the presence of succinate and in the presence of glutamate+malate+rotenone than human parahippocampal mitochondria (black bars). An explanation for this phenomenon is the mentioned higher specific electron transport activity of rat brain mitochondria in respect to human parahippocampal mitochondria (Fig. 4B). Really, as shown in detail in Table I, the electron transport chain of both types of brain mitochondria produces the by-product H₂O₂ at approximately similar percentage per consumed O₂ (with similar substrates), about 0.2% with glutamate+malate and about 1.5% with succinate.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Experimental traces of oxygen consumption of brain mitochondria oxidizing glutamate and malate. Air-equilibrated MTP medium at 30 °C with 10 mM glutamate, 5 mM malate and 3.3 mM MgCl2 was used. Thick traces, oxygen content (left axis); thin traces, rate of respiration (right axis). The numerical values at the thin curves represent averaged respiration rates (in nmol O2/min/mg protein) in the different mitochondrial states. Additions: ADP (250 µM), TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) (0.4 µM). A, rat brain mitochondria (RBM, 0.2 mg protein/ml). B, human brain mitochondria (HBM, 0.32 mg protein/ml).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Comparison of H2O2 production rate and maximal O2 consumption rate of rat and human brain mitochondria. A, H2O2 production rates: white bars, rat brain mitochondria (average protein concentration 0.17 mg protein/ml, n = 10); SUCC, 10 mM succinate; GLU+MAL+ROT, 10 mM glutamate + 5 mM malate + 6.7 µM rotenone; black bars, human brain mitochondria (average protein concentration 0.1 mg protein/ml, n = 8). The rates were determined at 30 °C in oxygen saturated MTP medium. B, O2 consumption rates: white bars, rat brain mitochondria (average protein concentration 0.2 mg protein/ml, n = 16); SUCC,10 mM succinate + 6.7 µM rotenone; GLU+MAL, 10 mM glutamate + 5 mM malate; black bars; human brain mitochondria (average protein concentration 0.35 mg protein/ml, n = 8). The maximal O2 consumption was induced by addition of 250 µM ADP and measured at 30 °C in MTP medium in the presence of 3.3 mM MgCl2.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Redox titration of mitochondrial superoxide production. Mitochondrial inner membrane preparations were incubated at about 0.7 mg/ml in oxygen saturated MTP medium in the presence of 6.7 µM rotenone. The -dependent oxidation of epinephrine to adrenochrome at different NADH/NAD+ ratios was followed spectrophotometrically at 486–575 nm with a dual wave-length spectrophotometer (open circles, conditions of measurement as described under "Experimental Procedures"). In control experiments (filled circles) we monitored the change in fluorescence of p-hydroxyphenylacetic acid (ex = 317 nm, em = 414 nm) catalyzed by horseradish peroxidase in the presence of 22 units/ml Cu,Zn-superoxide dismutase. The data were normalized in respect to the -generation rate in the presence of 1 mM NADH. For the calculation of redox potentials the midpoint potential of the free NADH/NAD+ couple Em 7.4 = –310 mV (21) and the Nernst equation were used.

Treatment of Rat Brain Mitochondria with H₂O₂ Increases the Rate of H₂O₂ Generation by Direct Electron Flow through Complex I Creating a Vicious Cycle—Because H₂O₂ has been reported to accumulate in the injured brain (22), which has been postulated to cause further injury accompanied by neuronal cell death, we investigated in further experiments how treatment of isolated rat brain mitochondria with H₂O₂ would affect the generation of H₂O₂. In these experiments isolated brain mitochondria were incubated for 10 min with H₂O₂ in a concentration range of 1 to 100 mM. After removal of H₂O₂ by re-sedimentation of mitochondria, we determined the rates of H₂O₂ production in the presence of the respiratory substrates glutamate+malate (Fig. 6A, filled circles), succinate (Fig. 6A, open circles), and in the presence of glutamate+malate+ rotenone (Fig. 6B, filled circles). As shown in Fig. 6A, 10 min incubation of rat brain mitochondria with concentrations of H₂O₂ in the range of 1–10 mM increased the generation of H₂O₂ with glutamate+malate as substrates (filled circles) but decreased succinate-dependent H₂O₂ production (open circles). The generation of H₂O₂ in the presence of glutamate+malate+ rotenone (Fig. 6B, filled circles) remained in this concentration range within experimental error nearly constant. To elucidate the potential cause for the increase of H₂O₂ generation by direct electron flow but the inhibition of H₂O₂ generation by reverse electron flow we determined the maximal oxygen consumption rates of H₂O₂-treated mitochondria in the presence of glutamate+malate (Fig. 6C, filled circles) and succinate+ rotenone (Fig. 6C, open circles). Very clearly, small amounts of H₂O₂ effectively inhibited maximal respiration with the complex I-dependent substrates glutamate+malate leaving the succinate+rotenone-dependent oxidation rate nearly unaffected. The almost negligible effect of H₂O₂ treatment in the 1–10 mM concentration range on mitochondrial oxidative phosphorylation with succinate (+rotenone) as substrate is further corroborated by the observation that the respiratory control ratio of mitochondria (RCR, defined as ratio between rates in active state (respiration rate in the presence of ADP) and resting state (respiration rate in the presence of ATP)) with this particular substrate changed from 2.7 ± 0.3 (control incubation) to 2.2 ± 0.2 (at 1 mM H₂O₂), to 2.1 ± 0.1 (at 5 mM H₂O₂) and to 2.1 ± 0.3 (at 20 mM H₂O₂). Only the incubation of mitochondria with concentrations of H₂O₂ above 50 mM seriously affected the coupling of mitochondrial oxidative phosphorylation: at 50 mM H₂O₂ the RCR declined to 1.7 ± 0.3, at 80 mM H₂O₂ to 1.2 ± 0.2, and at 100 mM the RCR was equal to 1 (fully uncoupled condition, four independent experiments).

View larger version (32K): [in this window] [in a new window]   FIG. 6. Influence of H2O2 treatment on H2O2 production (A and B), O2 consumption in active state (C), reduced glutathione content (D), and aconitase activity (E) of rat brain mitochondria. Mitochondria (four independent preparations) were suspended at about 3.5 mg protein/ml in MTP medium and treated with the indicated concentrations of hydrogen peroxide for 10 min. Thereafter, we centrifuged the suspension at 16,000 x g for 5 min. The resulting pellet was dissolved in MSE solution. In control experiments mitochondria were treated with equal quantities of water. A, H2O2 production (0.17 mg protein/ml) in oxygen saturated MTP medium in the presence of glutamate (10 mM) and malate (5 mM) (filled circles, solid curve) and succinate (10 mM) (open circles, dashed curve). B, H2O2 production in the presence of glutamate (10 mM) and malate (5 mM) and rotenone (6.7 µM). C, O2 consumption rate (0.2 mg protein/ml) in state 3 (after addition of 250 µM ADP) in the presence of glutamate (10 mM) and malate (5 mM) (filled circles, solid curve) or succinate (10 mM) and rotenone (6.7 µM) (open circles, dashed curve). D, GSH content; E, aconitase (filled circles, left ordinate axis) and citrate synthase (open circles, right ordinate axis) activities, assay conditions as described under "Experimental Procedures". The total GSH content (100%) of control mitochondria was determined to be 3.5 ± 1.5 nmol/mg protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the present study we have characterized the H₂O₂ generation of mitochondria isolated from rat brain and human parahippocampal cortex. In accordance with several previous reports (5, 12), but in clear contrast to others (6, 10), we observed that the main quantity of H₂O₂ is produced at a site within complex I of the respiratory chain whereas only rather small quantities (10–20% of maximal H₂O₂ generation) are produced at the bc₁ complex in the presence of antimycin A. One potential reason for the discrepant results could be the different quality of mitochondrial preparations (the specific rates of active state respiration of rat brain mitochondria with succinate as substrate reported in Ref. 6 are 10-fold lower than our rates). In accordance with previous work (19, 20) we detected a clear oxygen dependence of H₂O₂ generation with the rather high K_m value of 0.92 ± 0.01 mM oxygen (equal to 85% of maximal oxygen saturation at 30 °C) for all conditions studied. In line with these results, at 100% oxygen saturation the superoxide production of brain mitochondria leads to a diminished rotenone sensitivity of NADH/coenzyme Q₁ oxidoreductase. As shown for mitochondria from rat and human brain, under resting state conditions between 0.2% (with glutamate+malate) and 1.5% (with succinate) of respiratory chain electron transport is redirected to superoxide formation. The different total H₂O₂ generation rates observed for isolated rat and human brain mitochondria are most likely related to the different purity of the individual mitochondrial preparations.

Applying two different methods for superoxide detection we determined the redox potential of the superoxide producing site to be –295 mV. This result can explain the strict dependence of superoxide production on the functional state of mitochondria reported in previous work (5, 6, 12, 24). Conditions leading to high NAD-reduction (resting succinate respiration, inhibition of complex I by rotenone) stimulate superoxide production, whereas conditions that cause rapid oxidation of NADH (active state respiration or uncoupling) lead to very low superoxide production rates. Our result seems to be in contrast to the redox potential of –360 mV given in Ref. 12 for rat heart mitochondria. But for determination of the redox potential, these authors used the -hydroxybutyrate/acetoacetate redox couple in rat heart mitochondria, which contain only minor amounts of -hydroxybutyrate dehydrogenase not allowing an efficient substrate couple equilibration.

The following findings point to FMN as the potential site for superoxide generation. (i) The determined redox potential of –295 mV, which is in agreement with the midpoint potential of FMN but not with that of iron sulfur clusters.² (ii) The strong diphenyleneiodonium sensitivity (in agreement with Ref. 6) and the rather low p-chloromercuriobenzoate sensitivity (in contrast to Ref. 11) of this reaction. It is therefore very likely that the semireduced form of FMN-FMNH^· (in close similarity to the bound semiquinone QH^· of center i of bc₁ complex) is the competent one-electron donor for the one-electron reduction of oxygen to superoxide.

The following experimental problems can explain the controversial quantitative data for H₂O₂ and superoxide production of isolated mitochondria reported so far. (i) The rates of H₂O₂ generation are very low at 20% oxygen saturation used in the previous reports (5, 6, 10, 12, 24), which does not allow accurate determinations. (ii) Some of the fluorescent dyes (especially Amplex Red) tend to show high endogenous fluorescence changes for which considerable corrections have to be made (see discussion in Ref. 24). (iii) And finally, ketoacids, like pyruvate or oxoglutarate, tend to cause artificial fluorescence changes in the presence of p-hydroxyphenylacetate or Amplex Red, which can obscure accurate measurement of superoxide and H₂O₂ production. In the present study we therefore avoided the use of ketoacids as mitochondrial substrates and used p-hydroxyphenylacetate, which did not show considerable endogenous fluorescence changes as obtained with the more sensitive dye Amplex Red (data not shown). To obtain reliable data for maximal H₂O₂ and superoxide generation rates we also used oxygen saturated media.

Furthermore, we were able to show that incubation of rat brain mitochondria with H₂O₂ can initiate a vicious cycle of H₂O₂ generation, which might be of importance for certain pathological conditions of the brain. In vivo, during the ischemia-reperfusion period, the rather stable membrane-permeable H₂O₂ can reach stationary concentrations of about 100 µM (22). In our in vitro experiments, we applied a short-term incubation (10 min) of rat brain mitochondria with H₂O₂ in a concentration range of 1–10 mM, which is 10–100-fold higher than the maximal concentrations observed in vivo (however, the in vivo challenge of mitochondria with the lower H₂O₂ concentrations might be of much longer duration). We observed increased H₂O₂ generation by direct electron transport and diminished H₂O₂ generation by reversed electron flow. On the other hand, the maximal oxygen consumption of these mitochondria was normal with succinate+rotenone but inhibited with glutamate+malate to about 50%, pointing to a selective inhibition of complex I activity. The H₂O₂ treatment in this concentration range did not affect the mitochondrial citrate synthase activity and only small alterations of respiratory control ratios with succinate (+rotenone) were observed. These findings exclude a possible unspecific disruption of mitochondrial function by the H₂O₂ treatment. Furthermore, it is improbable that this complex I inhibition is related to the recently reported glutathinylation of complex I (25), because under our conditions, the GSH levels were decreased only by about 30%. However, aconitase, which harbors a highly ROS-sensitive FeS cluster (23), was completely inhibited. Consequently, our findings allow the supposition of the scenario depicted in Fig. 7. H₂O₂ inactivates a ROS-sensitive FeS cluster within complex I localized between the coenzyme Q-reduction site and FMN (dotted line). This would initiate a vicious cycle leading to a self-accelerating inhibition of complex I with the result of further elevated H₂O₂ generation. This scenario might explain the following findings relevant to human disease: (i) the rather selective inhibition of complex I in combination with accumulation of markers for oxidative stress which has been reported for Substantia nigra in Parkinson's disease (26), and (ii) the detected inhibition of complex I in the epileptic focus of patients with temporal lobe epilepsy (27).

View larger version (18K): [in this window] [in a new window]   FIG. 7. Scheme illustrating the proposed vicious cycle of H2O2 effects on complex I of mitochondrial respiratory chain.

	FOOTNOTES

These authors contributed equally to this work.

^|| To whom correspondence should be addressed: Dept. of Epileptology, University of Bonn Medical Center, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Tel.: 49-228-287-5744; Fax: 49-228-287-9110; E-mail: wolfram.kunz{at}ukb.uni-bonn.de.

¹ The abbreviations used are: ROS, reactive oxygen species; FMN, flavine mononucleotide; GSH, reduced glutathione; CMB, p-chloromercuriobenzoate; RCR, respiratory control ratio.

² Complex I web site: www.scripps.edu/mem/biochem//CI/index.html.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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