Nonezymatic formation of succinate in mitochondria under oxidative stress

Nonezymatic formation of succinate in mitochondria under oxidative stress

Free Radical Biology & Medicine 41 (2006) 56 – 64 www.elsevier.com/locate/freeradbiomed Original Contribution Nonenzymatic formation of succinate in...

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Free Radical Biology & Medicine 41 (2006) 56 – 64 www.elsevier.com/locate/freeradbiomed

Original Contribution

Nonenzymatic formation of succinate in mitochondria under oxidative stress Nadezhda I. Fedotcheva a,⁎, Alexander P. Sokolov b , Mariya N. Kondrashova a a

b

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia Received 20 October 2005; revised 3 February 2006; accepted 18 February 2006 Available online 13 March 2006

Abstract The products of the reactions of mitochondrial 2-oxo acids with hydrogen peroxide and tert-butyl hydroperoxide (tert-BuOOH) were studied in a chemical system and in rat liver mitochondria. It was found by HPLC that the decarboxylation of α-ketoglutarate (KGL), pyruvate (PYR), and oxaloacetate (OA) by both oxidants results in the formation of succinate, acetate, and malonate, respectively. The two latter products do not metabolize in rat liver mitochondria, whereas succinate is actively oxidized, and its nonenzymatic formation from KGL may shunt the tricarboxylic acid (TCA) cycle upon inactivation of α-ketoglutarate dehydrogenase (KGDH) under oxidative stress, which is inherent in many diseases and aging. The occurrence of nonenzymatic oxidation of KGL in mitochondria was established by an increase in the CO2 and succinate levels in the presence of the oxidants and inhibitors of enzymatic oxidation. H2O2 and menadione as an inductor of reactive oxygen species (ROS) caused the formation of CO2 in the presence of sodium azide and the production of succinate, fumarate, and malate in the presence of rotenone. These substrates were also formed from KGL when mitochondria were incubated with tert-BuOOH at concentrations that completely inhibit KGDH. The nonenzymatic oxidation of KGL can support the TCA cycle under oxidative stress, provided that KGL is supplied via transamination. This is supported by the finding that the strong oxidant such as tert-BuOOH did not impair respiration and its sensitivity to the transaminase inhibitor aminooxyacetate when glutamate and malate were used as substrates. The appearance of two products, KGL and fumarate, also favors the involvement of transamination. Thus, upon oxidative stress, nonenzymatic decarboxylation of KGL and transamination switch the TCA cycle to the formation and oxidation of succinate. © 2006 Elsevier Inc. All rights reserved. Keywords: 2-Oxo acid; Succinate; Decarboxylation; Mitochondria; tert-butyl hydroperoxide; Hydrogen peroxide; Free radicals

Introduction Oxidative stress leads to the inhibition of some enzymes of the tricarboxylic acid (TCA) cycle. Aconitase is inhibited to the greatest extent up to complete inactivation at a H2O2 concentration of 50 μM [1,2,4]. Ketoglutarate dehydrogenase (KGDH) is partly inhibited at higher H2O2 concentrations. An approximately 40% decrease in the activity of KGDH was observed by the action of H2O2 at a concentration of 50– Abbreviations: KGL, 2-oxoglutarate; PYR, pyruvate; OA, oxaloacetate; GLU, glutamate; tert-BuOOH, tert-butyl hydroperoxide; TCA, tricarboxylic acid; MAL, malate; FUM, fumarate; ROS, reactive oxygen species; KGDH, αketoglutarate dehydrogenase; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; HPLC, high-performance liquid chromatography; HIF-1α, hypoxiainducible factor. ⁎ Corresponding author. Fax: +7 0967 79 05 33. E-mail address: [email protected] (N.I. Fedotcheva). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.02.012

500 μM [2] and the product of lipid peroxidation 4-hydroxy-3nonenal at a concentration of 50 μM [3]. Treatment of mitochondria with 500 μM tert-BuOOH resulted in a 90% decrease of KGDH activity [5]. Succinate dehydrogenase is not affected by H2O2 and tert-BuOOH [2,5]. It is known that the mechanism of inhibition of KGDH is related to the modification of its thiol groups by ROS [3,4,10]. The decrease in the activity of KGDH was observed in a number of diseases, some of which are assumed to be caused by oxidative stress and others, by mutations or the deficiency of the enzyme [6–9]. It was shown that the inactivation of KGDH leads to the disturbance of the TCA cycle, a decrease in the cell antioxidative status due to the dihydrolipoamide-dependent reduction of the key cellular antioxidant thioredoxin, and a decrease in ATP synthesis [6–9]. It was established that KGDH is not only a target of ROS but also their products under the conditions of a deficit of oxidized NAD+ and thioredoxin [10,11].

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It is known that 2-oxo acids are oxidized nonenzymatically by hydrogen peroxide to release CO2 and reduce H2O2 to water [12–15]. This reaction underlies the cytoprotective effect of 2-oxo acids, in particular pyruvate, observed in cell cultures after the addition or endogenous generation of H2O2 [13,16]. Pyruvate and other 2-oxo acids also protected against oxidative damage induced by tert-BuOOH, and the most probable mechanism of the protection is the scavenging of tert-BuOOH. [15]. We suggested that the oxidative decarboxylation of KGL by H2O2 not only lowers the level of ROS but also compensates for the inhibition of the enzymatic oxidation of KGL. According to the mechanism of oxidative decarboxylation of 2-oxo acids by H2O2, R-CO-COOH + H2O2 = R-COOH + CO2 + H2O [16], the reaction of KGL with H2O2 should result in the formation of succinate, which would support the TCA cycle. Acetate was formed from pyruvate in the reaction with peroxynitrite; in this case, nitrosocarbonate is produced instead of CO2 [17–19]. Presumably, the interaction of OA with H2O2 by this pathway should lead to the formation of malonate. To verify the suggestion that the nonenzymatic oxidation of KGL plays a compensatory role, we studied the products of oxidation of KGL and other mitochondrial 2-oxo acids by H2O2 and tert-BuOOH and determined whether this process can occur in mitochondria. Another goal of the work was to examine whether KGL enters the TCA cycle through the transamination under oxidative stress. In this case, the initial steps of the Krebs cycle are bypassed, and the switching to the truncated Krebs cycle occurs, which is typical for tumor cells [20,21]. The shortening of the Krebs cycle and its acceleration were also shown upon activation of physiological functions by adrenaline [22,23]. This shunt increases the contribution of succinate to the total oxidation, which provides acceleration of the energy supply. Tretter and Adam-Vizi [24] showed that the formation of KGL from glutamate activates the TCA cycle if KGDH is only partly inhibited by H2O2. We found that transamination supports the high rate of respiration in the presence of high tert-BuOOH concentrations completely blocking KGDH. Experimental procedures Wistar male rats weighing 200–220 g were used. Liver mitochondria were isolated by differential centrifugation; the isolation medium contained 300 mM sucrose, 10 mM Tris, pH 7.5. Protein concentration in a mitochondrial suspension was 74 mg/ml. Respiration of mitochondria was measured by the polarographic method in a 1-ml cuvette with stirring and thermostating at 26°C. The incubation medium contained 125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.4. Simultaneously the computer-assisted registration of CO2 was performed using a pCO2-selective electrode (Radiometer, Copenhagen). The products of oxidation of 2-oxo acids were analyzed by HPLC. The concentration of the carboxylic acids was determined on an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad). Elution was with H2SO4 (4 mM) at 35°C; the elution rate was 0.6 ml/min. Eluted acids were detected at 210 nm. The

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calibration curves were constructed using the standard solutions of respective authentic acids. For the analysis of the reaction products in a buffer solution, 2oxo acids were incubated with H2O2 or t-BuOOH in a buffer containing 125 mM KCl, 10 mM Hepes, pH 7.5, in a 1-ml polarographic cuvette equipped with O2- and CO2-selective electrodes. The incubation was carried out for 2–10 min, which corresponded to the complete release of CO2 in each separate sample, registered by a CO2-selective electrode. After the termination of the reaction, samples were taken for the analysis of the products. For the analysis of the products of KGL oxidation in mitochondria the duration of incubation with oxidants was increased to 60 min to raise the yield of the products. Each sample contained 2 ml of buffer (125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.4) and 100 μl of liver mitochondria (7.4 mg of protein). Mitochondria were incubated with KGL and oxidants in open cuvettes with continuous stirring. The aliquots of reaction mixture (0.2 ml) were mixed with 0.2 ml of 6% HClO4 and the denatured proteins were removed by centrifugation for 10 min at 14,000g. The supernatant was used for the analysis of products. For the analysis of transamination products, mitochondria were incubated with 4 mM GLU and 4 mM MAL in the presence of the uncoupler and varying concentrations of tert-BuOOH until complete depletion of oxygen in a closed polarographic cuvette. After the termination of respiration, samples for HPLC of the products were prepared as described above. 2-Oxo acids, carboxylic and dicarboxylic acids, buffers, tertBuOOH, and hydrogen peroxide were from Sigma (USA). Results Decarboxylation of 2-oxo acids by hydrogen peroxide and t-BuOOH in solutions Fig. 1 shows the decarboxylation of mitochondrial 2-oxo acids by H2O2, which was estimated by measuring the level of CO2. It is evident that the rate of decarboxylation of pyruvate by H2O2 was considerably higher than that of KGL and OA (Figs. 1, a, b, and c). The addition of catalase inhibited the formation of CO2 (Fig. 1, d), which confirms the complete dependence of the reaction on H2O2. Similar to H2O2, but at a concentration one order of magnitude higher, tert-BuOOH led to the decarboxylation of 2-oxo acids (Fig. 2). The reactions of 2-oxo acids with tertBuOOH were not inhibited by catalase and are, consequently, not related to the possible presence or formation of H2O2. The results of chromatographic analysis of the reaction products are presented in Fig. 3. It is seen that, in the reaction with both H2O2 and tert-BuOOH, 2-oxoglutarate, oxalacetate, and pyruvate are oxidized to succinate, malonate, and acetate, respectively. As the concentration of H2O2 increases from 2 to 10 mM, the amount of the substrate markedly decreases, and the yield of the product increases (Figs. 3A, B, and C). H2O2 is much more effective than tert-BuOOH (Fig. 3D). The product

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Fig. 1. Nonenzymatic decarboxylation of 2-oxo acids by H2O2. The concentration of 2-oxo acid 4 mM: (a) pyruvate, (b) OA, (c) KGL, (d) pyruvate + catalase.

concentrations were 2.5, 3.4, and 3.1 mM for succinate, acetate, and malonate, respectively, at 5 mM H2O2 and the duration of the reaction for 2 min, while similar yields of the products with tert-BuOOH were achieved at a concentration of 20 mM after a 60-min incubation. Nonenzymatic oxidation of KGL in mitochondria Nonenzymatic oxidation of KGL is of greatest interest as compared to other 2-oxo acids since it proceeds with the formation of succinate, which can shunt ROS-inactivated KGDH and the first complex of the respiratory chain. The nonenzymatic oxidation of KGL in mitochondria by H2O2 and tert-BuOOH was determined by the level of CO2, succinate, and the products of succinate oxidation, fumarate and malate. Fig. 4 shows the effect of hydrogen peroxide on O2 consumption and CO2 yield in the course of uncoupled oxidation of KGL. Upon inhibition of respiration by rotenone, the addition of 500 μM H2O2 resulted in the release of oxygen to 200 μM, indicating that practically all H2O2 added was converted by catalase to oxygen. In order to maintain the H2O2 level, sodium azide was added. As noted above, rotenone was added to inhibit the enzymatic oxidation of KGL. These inhibitors completely blocked respiration, i.e.,

Fig. 2. Nonenzymatic decarboxylation of 2-oxo acids by tert-BuOOH. Black curves, formation of CO2 in the absence of catalase; gray curves, formation of CO2 in the presence of catalase. Additions: pyruvate 10 mM, tert-BuOOH 20 mM, H2O2 0.5 mM.

Fig. 3. HPLC of the products of nonenzymatic decarboxylation of 2-oxo acids by H2O2 and tert-BuOOH. Reaction mixtures of 5 mM 2-oxo acid with 2 mM (A), 5 mM (B), 10 mM (C) H2O2 after a 2- to 10-min incubation, which corresponded to the complete release of CO2 in each separate sample, registered by a CO2-selective electrode, and with 20 mM tert-BuOOH (D) after a 1-h incubation. The retention time of the substrate and the product of each reaction are indicated. The retention time of the standards is shown on curve E: KGL 8.29 min, PYR 9.25 min, OA 7.91 min, succinate (SUC) 11.89 min, acetate (AC) 15.22 min, and malonate (MLN) 9.93 min. Since the absorption coefficients for 2-oxo acids are about one order of magnitude higher than those for the other carboxylic acids, the signals from the products were magnified 8-fold.

the enzymatic oxidation of KGL and enzymatic formation of CO2. In the presence of these inhibitors, further addition of H2O2 resulted in the formation of CO2, indicating the nonenzymatic decarboxylation. The rate of KGL nonenzymatic decarboxylation by 0.5 mM H2O2 was 190 μM CO2 per minute, which is close to the rate of enzymatic decarboxylation in the presence of the uncoupler (120 μM CO2/min). Nonenzymatic oxidation of KGL in mitochondria also occurred in the presence of menadione. Menadione, 2-methyl1,4-naphthoquinone, induces the generation of H2O2 through the redox-cycling process that involves reductases and diaphorases [25–27]. Menadione strongly activated the respiration and the release of CO2 in the presence of KGL and rotenone (Fig. 5). The activation of respiration in this

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Fig. 4. Effect of H2O2 on CO2 formation from KGL in mitochondria in the presence of inhibitors of KGL enzymatic oxidation. Additions: KGL 4 mM, CCCP 1 μM, rotenone 2.5 μM, sodium azide 1 mM, and H2O2 0.5 mM; rat liver mitochondria 3.5 mg protein. The incubation medium contained 125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.4.

case may be related to two factors: the diaphorase activity of dihydrolipoamide dehydrogenase [28,29] and the nonenzymatic oxidation caused by the generation of ROS. As judged from the inhibition by malonate, the 50% oxygen uptake in the presence of menadione is due to the oxidation of succinate. Malonate does not inhibit the CO2 release since it is caused by KGL oxidation only. If rotenone was used in combination with azide, the oxygen uptake and CO2 release decreased but were still detected. The azide-insensitive fraction accounts for 30% of the control, as judged by the O2 uptake, and 70%, as judged by the CO2 release per se. Most probably, all CO2 formed in the presence of sodium azide is the product of the nonenzymatic decarboxylation of KGL. As for the O2 uptake, the results obtained are consistent with the reported data indicating that cyanide-resistant respiration in the presence of menadione varies from 15 to 60% [27]. The addition of tert-BuOOH at a concentration of 500 μM led to the complete inhibition of respiration in the presence of KGL (Fig. 6). Respiration was not activated by menadione and

Fig. 6. Effect of tert-BuOOH on the respiration with KGL. Additions: KGL 4 mM, tert-BuOOH 0.5 mM, CCCP 1 μM, menadione 25 μM, succinate 4 mM; rat liver mitochondria 3.5 mg protein. The incubation medium contained 125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.4.

was strongly stimulated by succinate. The absence of the effect of menadione in this case, in contrast to its activating effect in the presence of rotenone (Fig. 5), is evidence that tert-BuOOH inhibits KGDH itself. Determination of TCA cycle substrates during the oxidation of KGL in the presence of oxidants Table 1 gives the concentrations of KGL oxidation products in mitochondria incubated with H2O2, menadione, and tertBuOOH. The measurements indicated that 5 mM KGL and 60min incubation were sufficient to determine all substrates of the TCA cycle formed during KGL oxidation: succinate, fumarate, and malate. If mitochondria were incubated without KGL,

Table 1 Distribution of Krebs cycle substrates upon oxidation of KGL in mitochondria in norm and during incubation with H2O2, tert-BuOOH, menadione, and inhibitors of enzymatic oxidation 5 mM KGL + additions

KGL oxidation products, mM Succinate

Fig. 5. Respiration and CO2 formation in mitochondria in the presence of inhibitors of KGL enzymatic oxidation and menadione. The rates of respiration and CO2 release in the presence of uncoupler are taken as 100%. Additions: KGL 4 mM, CCCP 1 μM, rotenone (Rot) 2.5 μM, menadione (Men) 25 μM, malonate (Mal) 4 mM, sodium azide (Az) 1 mM; rat liver mitochondria 3.5 mg protein. The incubation medium contained 125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.4.

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− Malonate 4 mM H2O2 0.1 mM* H2O2 + malonate Menadione 25 μM Menadione + malonate Rotenone 2,5 μM Rotenone + H2O2 0.1 mM a Rotenone + menadione Rotenone + menadione + malonate tert-BuOOH 0.5 mM tert-BuOOH + malonate tert-BuOOH + menadione Without KGL + 5 mM succinate

0.33 ± 0.02 0.91 ± 0.1 0.32 ± 0.02 0.81 ± 0.06 0.44 ± 0.03 0.84 ± 0.07 0.11 ± 0.05 0.65 ± 0.23 0.36 ± 0.08 0.87 ± 0.27

Fumarate 0.072 ± 0.02 0.015 ± 0.001 0.084 ± 0.04 0.027 ± 002 0.102 ± 0.02 0.015 ± 0.002 0.017 ± 0.008 0.133 ± 0.015 0.041 ± 0.01 0.005 ± 0.002

0.45 ± 0.03 0.006 ± 0.0005 0.57 ± 0.06 0.0006 ± 0.0003 0.56 ± 0.06 0.0017 ± 0.0006 − 0.3 ± 0.04

Malate 1.2 ± 0.2 − 2.0 ± 0.4 − 1.6 ± 0.3 − 0.25 ± 0.11 0.85 ± 0.17 0.57 ± 0.02 − 0.32 ± 0.06 − − 3.4 ± 0.3

Mitochondria were incubated with KGL, oxidants, and inhibitors in 125 mM KCl, 10 mM HEPES, pH 7.5, for 60 min in an open cuvette with stirring. Then 0.2-ml aliquots were taken, 0.2 ml of 6% HClO4 was added, the residue was removed by centrifugation at 14,000g, and the supernatant was used for HPLC of KGL products. a Ten additions in the course of incubation.

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oxidation of succinate leads to the formation of fumarate and malate, and their concentrations are 4- and 2-fold, respectively, higher than during KGL oxidation. Effect of tert-BuOOH on respiration supported by transamination

Fig. 7. Effect of tert-BuOOH on respiration supported by transamination. The rate of respiration with 4 mM GLU and 4 mM MAL in the presence of uncoupler is taken as 100%. Additions: CCCP 1 μM, rotenone 2.5 μM, malonate 4 mM, AOA 2 mM; rat liver mitochondria 3.5 mg protein. The incubation medium contained 125 mM KCl, 10 mM Hepes, 1.5 mM phosphate, pH 7.4.

endogenous substrates were not detected; consequently, they do not affect the determination of product concentrations. To prevent the oxidation of succinate as the product of either enzymatic or nonenzymatic oxidation of KGL, malonate was added. The addition of malonate resulted in the accumulation of succinate, a decrease in fumarate concentration by an order of magnitude and more, and the elimination of malate. In the upper part of Table 1, the data are presented on the effect of H2O2 and menadione on the yield of succinate, fumarate, and malate in the absence and presence malonate. In all samples, malonate increased the concentration of succinate about 2- to 2.5-fold. The addition of oxidants did not inhibit the KGL oxidation, as evidenced by a high level of succinate in the presence of malonate. The yield of malate was even higher than without oxidants. The concentration of fumarate is substantially lower in absolute value than that of succinate and malate. Nevertheless, the drastic changes in the fumarate level are another convincing evidence that the metabolites flow from KGL to malate, and this flow is substantially activated by H2O2 (by 17%) and menadione (by 41%). In the lower part of Table 1, the concentrations of succinate, fumarate, and malate are given in the course of the incubation of mitochondria with rotenone, which blocks the enzymatic oxidation of KGL. It is evident that their concentrations abruptly decreased. However, both H2O2 and menadione activated the formation of succinate, fumarate, and malate, which indicates the nonenzymatic oxidation of KGL. The nonenzymatic decarboxylation of KGL was well pronounced in the presence of tert-BuOOH. Although tertBuOOH completely blocked the enzymatic oxidation of KGL (Fig. 6), succinate was formed, and its concentration increased during the incubation with malonate. Fumarate and malate were also identified in these samples, which confirms the oxidation of KGL. Menadione did not increase their concentrations, just as it produced no effect on respiration in the presence of tert-BuOOH. In the last part of the table, the concentrations of KGL and succinate oxidation products are compared. It is evident that the

The data presented above indicate the occurrence of nonenzymatic oxidation of KGL in mitochondria in the presence of oxidants. We suggested that this process supports the TCA cycle under oxidative stress, provided that KGL is supplied via transamination. We found that transamination is highly resistant to such a strong oxidant as tert-BuOOH. Fig. 7 shows that after the simultaneous addition of transamination substrates, GLU and MAL, tert-BuOOH even at very high concentrations (500–1000 μM) did not inhibit respiration. The inhibition was observed only after the concentration of tertBuOOH was increased to 5000 μM. By contrast, the oxidation of MAL and GLU separately, as well as the oxidation of KGL, was completely inhibited even at a concentration of tertBuOOH of 0.5 mM. Interestingly, the strong decrease in the sensitivity of respiration to tert-BuOOH by simultaneous addition of GLU and MAL was also reported earlier; however, its mechanism was not considered [5]. As is shown in Fig. 7, the respiration with GLU and MAL in the presence of 1.0 mM tertBuOOH was inhibited by AOA, a transaminase inhibitor, as well as rotenone and malonate. The sensitivity to these inhibitors implies that transamination with subsequent formation and oxidation of KGL is involved in respiration. We studied the formation of the products of these processes. Identification of transamination products formed in the presence of tert-BuOOH The data on the effect of tert-BuOOH on the formation of TCA cycle substrates from GLU and MAL are summarized in Table 2. The presence of two products, KGL and fumarate, was established with confidence, indicating the involvement of transamination with the formation and subsequent oxidation of KGL. As the concentration of tert-BuOOH was increased to 5000 μM, the concentration of KGL increased, while the level of fumarate initially rose and then diminished. Consequently, Table 2 Effect of tert-BuOOH on the formation of Krebs cycle substrates upon respiration supported by transamination Glutamate 4 mM + malate 4 mM

Products of transamination and oxidation KGL (mM)

Fumarate (mM)

−tert-BuOOH +tert-BuOOH 1 mM +tert-BuOOH 5 mM

0.17 ± 0.02 0.24 ± 0.07 0.51 ± 0.03

0.22 ± 0.02 0.34 ± 0.08 0.31 ± 0.07

Mitochondria were incubated with glutamate and malate and varying concentrations of tert-BuOOH in 125 mM KCl, 10 mM Hepes, pH 7.5 in the presence of 1 μM CCCP until complete depletion of oxygen in a closed polarographic cuvette. Then 0.2-ml aliquots were taken, 0.2 ml of 6% HClO4 was added, the residue was removed by centrifugation at 14,000g, and the supernatant was used for HPLC of products.

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even high concentrations of tert-BuOOH do not inhibit the formation of KGL but only begin to suppress its oxidation. Thus, respiration supported by transamination is extremely resistant to tert-BuOOH. Discussion Nonenzymatic decarboxylation of 2-oxo acids under oxidative stress The interaction of 2-oxo acids with H2O2 occurs by oxidative decarboxylation linked with the reduction of H2O2 to water and the formation of CO2 and the respective carboxylic acid. The inhibition of CO2 release by catalase is evidence that the reaction completely depends on the hydrogen peroxide. The analysis of the reaction products by HPLC indicated that, in the presence of hydrogen peroxide, succinate is formed from KGL, malonate is formed from OA, and acetate is formed from PYR. The reaction of tert-BuOOH with 2-oxo acids results in the formation of the same products as on oxidation by hydrogen peroxide. Since catalase does not affect this reaction, it may be suggested that 2-oxo acids directly interact with tert-BuOOH. The nonenzymatic oxidation of 2-oxo acids in mitochondria can be revealed by several ways. In our experiments, the decarboxylation of KGL by hydrogen peroxide was clearly seen from the release of CO2 upon the inhibition of both respiration and catalase activity. Under these conditions, the formation of succinate is mainly linked with the nonenzymatic oxidation of KGL. This pathway is particularly clearly seen in the presence of rotenone and high concentrations of H2O2, i.e., under conditions close to those in which KGL interacts with hydrogen peroxide in buffer solutions. The data obtained suggest that KGL can be oxidized in mitochondria via the nonenzymatic process. That this process is activated by the metabolites of oxidative stress makes probable its occurrence in some pathological conditions. The oxidative decarboxylation of KGL by hydrogen peroxide may be important just in case ROS induced inhibition of KGDH, since it provides succinate to bypass the blocked site in the TCA cycle. At concentrations of hydrogen peroxide of about 100 μM, which corresponds to its level in some pathologies [12], the intensity of this process in mitochondria is comparable to that of the enzymatic oxidative decarboxylation. Transamination as a source of KGL for nonenzymatic oxidation Then the question arises as to the source of KGL for this reaction since the previous enzyme aconitase is extremely sensitive to hydrogen peroxide and is inactivated even at lower H2O2 concentrations than KGDH. A possible source of KGL might be the transamination of GLU with OA on the assumption that transaminase is not inactivated under oxidative stress. We found that transamination is remarkably resistant to oxidants. Consequently, this pathway can operate under oxidative stress. An even more interesting conclusion that

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emerges from our results is that not only transaminase but also KGDH, when interacting with endogenous KGL formed by transamination, exhibit a strikingly high stability to oxidants. Since KGL formed by transamination can be oxidized either enzymatically by KGDH or nonenzymatically by ROS, the inhibition of respiration by rotenone is evidence of the involvement of the enzymatic pathway. Therefore, not only transaminase but also KGDH, which interacts with endogenous KGL generated by transamination, are highly resistant to oxidants. This finding can be explained in terms of the structural organization of the multienzyme complex that combines dehydrogenases and aspartate aminotransferases, the so-called metabolon [30,31]. A spatial reconstitution of metabolon, performed on the basis of biochemical studies, indicated that KGDH is the center of this complex. It was shown that reactions accomplished by the complex occur with a much higher rate than reactions accomplished by separate enzymes [30,31]. Presumably, internal KGDH, as a component of the transaminase complex in intact mitochondria, is much more stable to oxidants than external KGDH interacting with the added KGL in swollen mitochondria. To summarize, two processes, nonenzymatic decarboxylation of KGL and transamination, switch the TCA cycle to the formation and oxidation of succinate. Nonenzymatic decarboxylation not only decreases the concentration of H2O2, as it was reported earlier [14–16], but also forms succinate, bypassing the NAD-dependent oxidation, and thereby supports the respiration. Transamination supplies KGL and reduces the sensitivity of NAD-dependent oxidation to ROS. Role of 2-oxo acid oxidation by ROS in TCA cycle and general metabolism A possible involvement of 2-oxo acid decarboxylation by hydrogen peroxide in the regulation of metabolism and intracellular signaling is visualized in Fig. 8. Fig. 8A gives a traditional scheme of the Krebs cycle and its modifications according to the literature data and the results of this work. The upper part shows bypass of the citric acid steps transformation by ROS and transamination. Under these conditions, the truncated Krebs cycle is realized. Instead of the complete sequence, the cycle is closed by transamination of OA with GLU, which leads to the formation of KGL and ASP. Being a part of the malate/aspartate shunt, these substrates enter the cytosol where OA is formed by transaminase or by citrate lyase. The nonenzymatic oxidation of KGL and OA can occur in both mitochondria and cytosol, which can be accompanied by a decrease in the synthesis of fatty acids, cholesterol, and glucose and the accumulation of the products of nonenzymatic oxidation: acetate, malonate, and succinate. Role of products of 2-oxo acid oxidation by ROS in physiological and pathological processes Although acetate is not a source of acetyl-CoA in animals, the enzymatic ligation for the production of acetyl-CoA from

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Fig. 8. Rearrangement of TCA cycle under H2O2 action. The appearance of products of 2-oxo acid nonenzymatic oxidation affecting metabolism (A) and signaling (B). (A) Regulation of metabolism. PYR, KGL, and OA are decarboxylated by hydrogen peroxide to form acetate, succinate, and malonate (broken line) instead of acetylCoA (acCoA), succinyl-CoA (sucCoA), and malate (MAL) or phosphoenolpyruvate (PEP) formed enzymatically (solid line) and used for the synthesis of fatty acids, cholesterol, and glucose. ROS inhibit the TCA cycle predominantly on the level of aconitase and KGDH (thick arrow). The truncated Krebs cycle instead of the omitted steps of citric acid (citrate, CIT, cis-aconitate, cAC, isocitrate, ISC) is closed by transamination of OA with GLU, which leads to the formation of KGL and aspartate (ASP). Being a part of the malate/aspartate shunt, these substrates enter the cytosol where OA is formed by transaminase from ASP (the right part of the figure) or citrate lyase from citrate (the left part of the figure). (B) HIF-1α stabilization and signaling function. HIF-1a degrades in cells at normal oxygen concentrations after prolyl residue hydroxylation by oxygen/KGL/Fe(II)-dependent hydroxylase. Hypoxia, succinate, and KGL decarboxylation by H2O2, which leads to a decrease in the KGL and an increase in the succinate levels, inhibits the enzyme, permitting the transport of HIF-1α to the nucleus and HIF-dependent transcription of a wide variety of genes responsible for oxygen transport, vascularization, and anaerobic energy production.

acetate and CoA is a key reaction in the catabolism of acetate formed by oxidation of ingested ethanol in the liver and recycling of acetylcholine in the nervous system [32]. When localized in the cytoplasm, acetyl-CoA synthetase provides acetyl-CoA for the synthesis of fatty acids and cholesterol (Fig. 8A). In contrast, mitochondrial acetyl-CoA synthetase produces acetyl-CoA for oxidation through the citric acid cycle in the heart and skeletal muscle of fasting animals [33]. The formation of malonate (malonic aciduria) is inherent in some pathological states such as cardiomyopathy, progressive encephalopathy, intermittent ketoacidosis, and hypoglycemia [34]. The biochemical disturbances underlying different variants of malonic aciduria are unknown. It is believed that one of the mechanisms is the deficiency of malonyl-coenzyme A decarboxylase, which catalyzes the conversion of malonyl-CoA to acetyl-CoA [35]. Presumably, the decarboxylation by hydrogen peroxide of OA, the key intermediate in transamination and gluconeogenesis processes, is the cause of malonic aciduria in pathologies associated with oxidative stress (Fig. 8A).

In mitochondria, malonate inhibits SDH, leading to the accumulation of succinate, similar to what is observed with the mutated enzyme [36]. Succinate has a strong regulatory effect in cytosol, where it is involved in the regulation of the transcription activity of the HIF-1α [36]. Hypoxia occurs during tumor formation, in diabetes and other ischemic diseases, as well as in normal tissue remodeling during pregnancy, lactation, muscle, and adipose tissue deposition. HIF-dependent transcripts are responsible for oxygen transport (erythropoiesis, neovascularization, iron metabolism), vascular tone (nitric oxide synthase and heme oxygenase), and anaerobic energy production (glucose uptake and glycolysis) [37,38]. HIF-1α is a target for degradation after prolyl residue hydroxylation by oxygen/KGL/Fe(II)-dependent hydroxylase at normal oxygen concentration in cells [38]. Both metabolites, KGL and succinate, participate in the regulation of transcription activity of HIF-1α, the former as a cosubstrate and the latter as an inhibitor of prolyl hydroxylase (Fig. 8B). Under oxidative stress, nonenzymatic decarboxylation of KGL by hydrogen

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peroxide decreases the KGL and increases the succinate levels, which can lead to the inhibition of hydroxylase, stabilization of HIF-1α, and hence the hypoxia-mimicking effect. Probably, it is this process that explains the stabilization of HIF-1α by ROS and, importantly, mainly by hydrogen peroxide rather than superoxide [39]. Our conclusion is favored by the finding that exogenous administration of H2O2 or t-butyl hydroperoxide stabilizes HIF-1α in normoxic cells [40]. Significance of 2-oxo acid decarboxylation by ROS in therapeutic strategy Formation of succinate by decarboxylation of KGL by H2O2 can be involved in several physiological and pathological processes. This process can occur both in cells and the intercellular matrix and liquids, which is important for the realization of the signaling function of succinate. The signaling (hormone-like) action of succinate, which manifests itself in increased release of adrenaline and stimulation of physiological functions after the administration into the organism in concentrations far below than what is necessary to supply mitochondria with substrate, has been demonstrated by many-year studies of our group [41–45] and is well explained by the presence of the specific receptor whose signal function is related to the expression of the stress hormone angiotensin [46]. Acknowledgment This research is supported by the Grant for Leading Scientific Schools 824.2003.4. References [1] Bulteau, A. L.; Ikeda-Saito, M.; Szweda, L. I. Redox-dependent modulation of aconitase activity in intact mitochondria. Biochemistry 42:14846–14855; 2003. [2] Nulton-Persson, A. C.; Szweda, L. I. Modulation of mitochondrial function by hydrogen peroxide. J. Biol. Chem. 276:23357–23361; 2001. [3] Humphries, K. M.; Szweda, L. I. Selective inactivation of alphaketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal. Biochemistry 37:15835–15841; 1998. [4] Nulton-Persson, A. C.; Starke, D. W.; Mieyal, J. J.; Szweda, L. I. Reversible inactivation of alpha-ketoglutarate dehydrogenase in response to alterations in the mitochondrial glutathione status. Biochemistry 42:4235–4242; 2003. [5] Rokutan, K.; Kawai, K.; Asada, K. Inactivation of 2-oxoglutarate dehydrogenase in rat liver mitochondria by its substrate and t-butyl hydroperoxide. J. Biochem. (Tokyo) 101:415–422; 1987. [6] Hong, Y. S.; Kerr, D. S.; Liu, T. C.; Lusk, M.; Powell, B. R.; Patel, M. S. Deficiency of dihydrolipoamide dehydrogenase due to two mutant alleles (E340K and G101del). Analysis of a family and prenatal testing. Biochim. Biophys. Acta 1362:160–168; 1997. [7] Saada, A.; Aptowitzer, I.; Link, G.; Elpeleg, O. N. ATP synthesis in lipoamide dehydrogenase deficiency. Biochem. Biophys. Res. Commun. 269:382–386; 2000. [8] Shany, E.; Saada, A.; Landau, D.; Shaag, A.; Hershkovitz, E.; Elpeleg, O. N. Lipoamide dehydrogenase deficiency due to a novel mutation in the interface domain. Biochem. Biophys. Res. Commun. 262:163–166; 1999.

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