Free Radical Research, June 2014; 48(6): 684–693 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.900175
ORIGINAL ARTICLE
Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production F. Scandroglio, V. Tórtora, R. Radi & L. Castro
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Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay Abstract The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC50 for aconitase inhibition ranging from 7 to 24 mM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient ( 0.95). Aconitase-specific activity varied widely among tissues examined from ~60 mU/mg in liver to 321 mU/mg in kidney at 21% O2. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O2 reflecting aconitase inactivation by oxygen-derived oxidants at 21% O2. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O2· and H2O2 formation by the respiratory chain. Keywords: aconitase 2, free radicals, hydrogen peroxide, metabolic control, mitochondria, superoxide radical Abbreviations: O2·, Superoxide radical; ·NO, nitric oxide; ONOO, peroxynitrite; CO3·, carbonate radical; H2O2, hydrogen peroxide; MRM, mitochondrial respiration medium; RCR, respiratory control ratio
Introduction Mitochondrial aconitase, an enzyme that catalyzes the reversible isomerization of citrate to isocitrate via cisaconitate in the citric acid cycle, contains an [4Fe-4S] prosthetic group in which one of the irons, Fea, is not ligated to a protein residue, and thus can bind to hydroxyl groups of substrates or water. Superoxide radical (O2·) and reactive species derived from its reaction with nitric oxide, ·NO (i.e., peroxynitrite, ONOO and carbonate radical, CO3·), rapidly react with the Fe-S cluster, yielding an inactive protein in vitro [1–6] that can be reactivated by reductants (such as glutathione) and iron in vivo. Nitric oxide also interacts directly and reversible with aconitase [1]; however, sustained ·NO slowly promotes total Fe-S cluster disassembly [3,7]. Proteomic analysis of mitochondria from animal models of sepsis, diabetes, and aging revealed aconitase inactivation and nitration in vivo [8–12], showing that mitochondrial aconitase is a main target of reactive species in vivo. Due to the specificity of O2· reaction toward [4Fe-4S] aconitase cluster, the relationship between inactive and active aconitase has been used as a method to measure steady-state O2· concentration in mitochondria [4,13].
Mitochondrial aconitase evolved from its a-proteobacterial ancestor aconitase B which is more sensitive to O2· than aconitase A that originated the cytosolic counterpart (cytosolic aconitase, also known as iron-responsive protein 1). The presence of an exquisitely O2·–sensitive enzyme within the mitochondrial matrix led to the speculation that mitochondrial aconitase may provide a redox control mechanism of the citric acid cycle and suggested that the electron transport chain could somehow control O2· production by mitochondria [14,15]. Though indirect evidence supports this idea [15], the role of aconitase as a redox rheostat has not been proved yet. Flux control theory determines the control that various steps in a pathway have over the global flux of that pathway [16]. Based on this theory, metabolic control analysis has been applied to the study of mitochondrial oxidative phosphorylation in order to explain and support tissue specificity in human mitochondrial diseases and the evidence that a reduction in the activities of some respiratory chain complexes is associated with aging or neurodegenerative diseases [16–19]. Mitochondrial respiratory complex proteins are present in excess of the amount required to meet normal metabolic demand; therefore, submaximal inhibition of the enzyme does not
Correspondence: Laura Castro, Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Avenida General Flores 2125, 11800 Montevideo, Uruguay. Tel: (598)-2924-3414 ext 3402. Fax: (598)-2924-9563. E-mail: [email protected] (Received date: 15 January 2014; Accepted date: 27 February 2014; Published online: 28 March 2014)
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affect the overall metabolic pathway until a threshold level of inhibition is reached, depending somehow on the control exerted that such enzyme has on the whole pathway. Also, metabolic control analysis afforded by citric acid enzymes over respiration has been used to characterize the impact of mitochondrial bioenergetics in dopaminergic cells [20]. Herein, using isolated mitochondria from different rat tissues with varied metabolic profiles, we determined the flux control coefficient elicited by aconitase over the citric acid cycle and the respiratory chain. We aimed to determine the threshold level of aconitase inhibition that produces metabolic impact and how aconitase inhibition impacts mitochondrial oxidant production, that is, O2· and hydrogen peroxide (H2O2). Metabolic control analysis was performed in order to assess the role of aconitase as a redox sensor to test the hypothesis that increased levels of mitochondrial oxidants will impact on aconitase activity, thereby diminishing the reducing equivalents entering into the electron transport chain thus stopping the propagation of free radical generation by mitochondria. Experimental Reagents Chemicals were supplied by Sigma Chemical Co. (St. Louis, MO, USA) or Applichem GmbH (Germany). Amplex red was obtained from Molecular Probes-Invitrogen (Eugene, OR, USA). Mitochondrial isolation Intact liver, kidney, hearts, and brain mitochondria were prepared by differential centrifugation as described previously [21] from adult Wistar rats (200–250 g). Liver mitochondria were isolated from overnight fasted animals. To obtain heart mitochondria, hearts were minced into small pieces with a tissue grinder before homogenization with the Potter-Elvehjem. Due to the purification procedure, brain mitochondria obtained in this study consist preferentially of non-synaptosomal mitochondria. All experiments were performed with the approval of the Institutional Animal Welfare Committee. Mitochondrial pellets were resuspended in a minimal volume of MRM buffer (0.3 M sucrose, 5 mM potassium phosphate, 5 mM morpholinepropanesulfonic acid, 1 mM EGTA, 0.1% bovine serum albumin, pH 7.4), and protein concentration was determined according to the Bradford method. In some experiments, mitochondria were exposed to three cycles of freeze–thaw in order to obtain broken mitochondria. Aconitase activity Aconitase activity was measured in fresh sonicated mitochondria (15 mg/ml) with the addition of citrate and followed by the appearance of cis-aconitate at 240 nm in 100 mM Tris–HCl of pH 7.4. Inhibitor titration using
increasing concentrations of fluorocitrate (0–60 mM) was performed. The concentration of fluorocitrate needed to inactivate 50% of aconitase activity (IC50) was calculated by adjusting the plot to a single exponential function. Maximal aconitase-specific activity found for the different tissues was determined after the incubation of fresh sonicated mitochondria for 30 min under very low oxygen tension ( 2% oxygen) in a Coy lab hypoxic chamber. Oxygen consumption measurements Oxygen consumption elicited by substrates was evaluated in fresh mitochondrial (0.01–0.05 mg/ml) preparations by high-resolution respirometry (Oroboros®, Insbruck, Austria) at 37°C, in MRM buffer. Calibration with airsaturated buffer was performed daily. Data were collected and analyzed using software DatLab (Oroboros) displaying real-time oxygen concentration and oxygen flux, that is, the negative-time derivative course of oxygen concentration. Complex I and Complex II-dependent respiration was routinely assessed using 10 mM glutamate/2 mM malate or 10 mM succinate. Respiratory control ratio was evaluated after the addition of 1 mM ADP, and residual oxygen consumption was determined by the addition of 5 mM antimycin-A. As citrate alone did not support respiration in our mitochondrial preparations, we evaluated aconitase through citric acid cycle-complex I-dependent respiration by adding 10 mM citrate/2 mM malate as substrates as previously reported [20]. In the latter conditions, titrations with fluorocitrate were performed during state 3 respiration. Aconitase-driven respiration was confirmed by recovered respiration afforded by 5 mM isocitrate after fluorocitrate titrations. Aconitase expression in different rat tissues Mitochondrial proteins (30 mg) from brain, heart, liver, and kidney were separated by 10% SDS–PAGE, and aconitase tissue levels were analyzed by Western blot using a polyclonal anti-aconitase 2 antibody from ABCAM. Immunoreactive proteins were detected and quantified using the Odissey Infrared Imaging System. Mitochondrial hydrogen peroxide production and membrane potential determination Mitochondrial H2O2 release arising from O2· dismutation [22] was measured in 0.1 mg protein/ml intact mitochondrial suspensions in MRM buffer at 37°C, with continuous stirring. Amplex Red (25 mM) oxidation was followed in the presence of 0.5 U/ml horseradish peroxidase and 10 mM citrate/2 mM malate in the presence of 1 mM ADP. In some conditions, fluorocitrate was added at two different concentrations: equal to threshold value and equal to IC50. Amplex Red is oxidized, generating resorufin, which can be detected fluorimetrically using a plate reader fluorescence spectrophotometer (Fluostar) lex 530 nm, lem 590 nm. Controls conducted in the absence of
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686 F. Scandroglio et al. mitochondria or in the absence of peroxidase indicate that nonspecific probe oxidation is negligible as reported previously [22]. As a positive control, antimycin A in the presence of substrates that triggers O2· formation by Complex III was used and resulted in 6–10 times higher H2O2 formation than control values. Calibration was performed by adding H2O2 at known concentrations (e240nm 43.6 M–1 cm–1). Mitochondrial ∆Y was estimated through fluorescence changes of 5 mM safranin O at lex 486 nm and lem 585 nm at 37°C, as described in [22,23]. For these purposes, mitochondria were isolated in potassium-free medium as described in [23]. Incubation conditions were the same as for H2O2 release measurements. Data obtained were calibrated using a K gradient. The ∆Y value obtained for each K concentration was determined using the Nernst equation, assuming intramitochondrial [K] to be 120 mM, and plotted against measured fluorescence values to generate a calibration curve for each tissue. Calculation of flux control coefficients and respiratory threshold Flux control coefficients were calculated according to the metabolic control theory [16]. Control coefficients quantitatively describe the control exerted by and enzyme in a metabolic step or network over substrate flux. The flux control coefficient can be defined as the fractional change in pathway flux of a metabolic network under steady-state conditions, induced by a fractional change in the individual step under consideration. A consequence of the definition of control coefficient is the summation theorem, stating that in a metabolic network, the sum of the flux control coefficient of each step of the network is equal to one. In this case, we are analyzing only one step (aconitase) of the entire pathway. For aconitase-dependent oxidative flux (respiration) in mitochondria:
dJO 2 d (Inhibitor) I CJ dVc d (Inhibitor)
(1)
where CIJ is the flux control coefficient of aconitase, dVc/d(Inhibitor) is the rate of change of aconitase activity (individual step), and dJO2/d(Inhibitor) is the rate of change of respiration (global flux), at low concentrations of the complex inhibitor. Fluorocitrate, a suicide aconitase inhibitor [24], was used to performed aconitase activity and aconitase-dependent respiration titrations in order to assess control coefficients and threshold values according to [20,25]. The threshold curves come from the titration curves. Each point of a threshold curve represents the respiratory rate inhibition percentage as a function of inhibition percentage of aconitase activity for the same fluorocitrate concentration.
General procedures All experiments reported herein were reproduced at least thrice, and results shown correspond to one representative experiment of each one or the mean SD. For statistical analysis, independent t-tests were performed, with a 0.05, P 0.05. Graphics and mathematical fits to experimental data were performed using OriginPro 8 (OriginLab Corporation). Results Fluorocitrate titration of aconitase in different rat tissues In order to perform a metabolic control analysis, the measurement of the impact of increasing concentration of specific inhibitors on enzyme activities versus substrate specific respiration is used to obtain titration curves for graphical determination of flux control coefficient, an index of enzyme contribution to flux control in the entire pathway. Therefore, it is necessary to evaluate aconitase enzyme activity as well as aconitase-driven NADH production and oxygen consumption. Aconitase catalytic activity and aconitase-dependent respiration were titrated with the specific inhibitor fluorocitrate both as isolated reaction step in sonicated mitochondria and as respiratory chain integrated steps in intact mitochondria, respectively. In this way, it was possible to determine at each concentration of fluorocitrate used, the percentage of inhibition of aconitase activity in permeabilized mitochondria and how this inhibition affects the respiratory flux. Figure 1 shows that increasing concentrations of fluorocitrate (0–40 mM) decreased aconitase-catalytic activity (A) and citratedependent respiration in kidney mitochondria (B). In respirometry experiments, after successive fluorocitrate additions, isocitrate addition resulted in recovery of oxygen consumption showing the specificity of aconitasedependent respiration in this assay (Figure 1B) and the integrity of the other steps of the citric acid cycle and electron transport chain. Possible effects of fluorocitrate in electron transport chain were also excluded by performing experiments with broken mitochondria in which NADH-dependent oxygen consumption rates were evaluated with or without fluorocitrate which showed similar rates (not shown). Similar plots were obtained for other tissues analyzed (heart, brain, and liver—not shown). The respiratory control ratio (RCR) calculated as the ratio of oxygen consumption rates after (state 3) and before (state 4) ADP addition using citrate as substrate was always lower than for glutamate/malate (RCR from 5 to 7) or succinate (RCR for from 3 to 5), probably reflecting citrate constraints to reach the mitochondrial matrix. The flux control coefficient (CIJ) for aconitase in the different organs analyzed was calculated as described earlier and gave the CIJ 0.54 for aconitase in kidney. The threshold level of aconitase inhibition over respiration was
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Aconitase inactivation and mitochondrial metabolic control 687
Figure 1. Fluorocitrate titrations of aconitase. (A) Aliquots of fresh sonicated kidney mitochondria are incubated with 100 mM citrate in 100 mM Tris–HCl, pH 7.4, in the absence or in the presence of increasing mM concentration of fluorocitrate as indicated in the right of panel A. Aconitase activity is measured following cis-aconitase formation at 240 nm. (B) A typical oxygen consumption trace for fresh kidney mitochondria preparations. Measurements are performed at 37°C in the 2-ml chamber of the Oroboros Oxygraph-2k in mitochondrial respiration medium. The following are then added: Mt, 0.01 mg/ml mitochondria; Malate/citrate, 10 mM citrate/2 mM malate; 1 mM ADP; 30 mM and 50 mM Fluorocitrate; 5 mM Isocitrate. Blue line: oxygen concentration (mM); red line: oxygen flow (pmol/sec.mg). Color available at the web version of this article.
determined from a curve that expressed the rate of oxygen consumption as a function of aconitase inhibition (Figure 2C) which came from combining y-axes of A and B plots in Figure 2. Kidney aconitase inhibition threshold was found to be 63%, beyond which respiration decreased rapidly. Supplementary Figure 1 available online at http:// informahealthcare.com/doi/abs/10.3109/10715762.2014. 900175 shows data obtained for brain, heart, and liver mitochondria. According to the threshold curves from the different tissues analyzed, two distinct types of behavior can be distinguished as previously reported [26]. For liver and kidney, a plateau is observed followed by a stepped breakage, whereas for the heart and brain threshold curves the plateau is not evident (Figure 2C and Supplementary Figure 1 available online at http://informahealthcare.com/ doi/abs/10.3109/10715762.2014.900175). Table I summarizes the data obtained for the different organs analyzed. Titrations with fluorocitrate showed tissue variations with IC50 of 24, 24, 7, and 12 mM in kidney, liver, brain, and heart, respectively (Table I). Brain tissue exhibited the highest flux control coefficient (0.98). Before significant changes in oxygen consump-
tion were observed, aconitase inhibition thresholds were determined to be 63, 69, 95, and 98% for kidney, liver, brain, and heart, respectively (Table I), meaning that in liver and kidney, ~40% inhibition of aconitase is required to slow respiration, but less than 5% inhibition of aconitase will impact on brain and 2% in heart mitochondrial respiration. Although aconitase is not considered to be a rate-limiting enzyme in the citric acid cycle, its inhibition could impact the generation of NADH by the citric acid cycle and its supply to electron transport chain as could be envisaged by flux control coefficient obtained, especially for brain and heart. Membrane potential and hydrogen peroxide production in aconitase-inhibited mitochondria As mitochondrial O2· and H2O2 production increases when there is abundance of reduced transport chain complexes which led to mitochondrial inner membrane hyperpolarization [27], we measured mitochondrial membrane potential in control and aconitase-inhibited mitochondria. As shown in Figure 3, H2O2 released by
688 F. Scandroglio et al. Table I. Comparative parameters obtained from rat tissues. SA (nmol/mg.ml) Tissue IC50 (mM) Kidney Liver Brain Heart
24 24 7 12
CIJ 0.54 0.018 0.13 0.008 0.98 0.023 0.80 0.015
Threshold 21% O2 63 69 95 98
2% O2
321 25 307 30 59.2 3 55 7 115 15 163 12* 296 13 334 14*
A, specific activity; CJI, flux control coefficient; IC50, fluorocitrate S concentration needed to inhibit aconitase activity by 50%; Threshold, level of aconitase inhibition needed to significantly affect respiration. *p 0.05 significantly to 21% O2.
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mitochondria from all tissues analyzed increased when citrate was present as substrate and an accompanying increase in ∆Y across inner mitochondrial membrane (compare first and second bars in graphs from left and right columns in Figure 3). Hydrogen peroxide generation rate varied significantly among the studied tissue. In the presence of citrate and ADP, H2O2 production rate was 10.6 0.8 nmol.min 1.mg 1 for kidney, 1.8 0.2 nmol.min 1.mg 1 for brain, 7.3 0.3 nmol. min 1.mg 1 for heart, and 15 0.3 nmol.min 1.mg 1 for liver mitochondria. These differences were in line with tissue variations reported for other substrates [22]. When fluorocitrate was added at a concentration which correlates to that obtained at threshold level when respiration is compromised, released H2O2 is decreased, and a drop in ∆Y was observed (Figure 3E–H). The effects of fluorocitrate were higher at the concentration that correlates with the obtained IC50 for each tissue. Interestingly, these effects were statistically significant in brain and heart, the tissues with the highest CIJ. Aconitase expression and maximal aconitase activity in different rat tissues
Figure 2. Measurement of mitochondrial respiratory threshold and calculation of flux control coefficient elicited by aconitase. (A) Fresh kidney mitochondria (15 mg/ml) are sonicated, and aconitase activity is immediately assayed using citrate as substrate and increasing concentrations of fluorocitrate (0–60 m M). Aconitase activity is expressed as percentage of the control. The concentration of fluorocitrate needed to inactivate 50% of aconitase activity (IC50) is calculated by adjusting the plot to a single exponential function. (B) Oxygen consumption in the presence of fluorocitrate is determined when the flux reached a steady state after each addition. Oxygen consumption (respiration) is expressed as percentage of citrate-dependent respiration in the presence of ADP (State 3). (C) Threshold curves from fluorocitrate titration are constructed by combining y-axis of the graphs A and B. Metabolic
Aconitase-specific activity varied significantly over the tissues studied herein (Table I). Mitochondrial aconitase is a constitutive protein, but the amount of the enzyme differed in the organs analyzed (Figure 4). As observed in the Western blot, though aconitase expression in brain and kidney was similar, specific activity was not. As it was previously reported, a fraction of inactive aconitase is found in basal condition which reflects O2· steadystate production in cells [4]. Therefore, we explored if the discrepancies observed between specific activity and expression, particularly for brain aconitase could be attributed to a pool of inactive aconitase present in this tissue. Incubation of sonicated mitochondria in a very low O2 atmosphere ( 2% O2) for 30 min yielded 42% higher brain aconitase-specific activity than that observed
control analysis is used to determine the flux control coefficient for aconitase considering the rate of change of oxygen consumption (entire flux) at low concentration of fluorocitrate and the rate of change of activity as explained in the text.
Aconitase inactivation and mitochondrial metabolic control 689
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Figure 3. Aconitase inhibition leads to a decrease in mitochondrial membrane potential and a diminished hydrogen peroxide production. (A–D) Hydrogen peroxide fluxes were measured in 0.1 mg/ml mitochondrial suspensions following the oxidation of Amplex red as described under the Experimental section. 100% correspond to: 10.6 0.8 nmol/mg.min for kidney, 1.8 0.2 nmol/mg.min for brain, 7.3 0.3 nmol/mg.min for heart, and 15 0.3 nmol/mg.min for liver mitochondria. (E–H) Mitochondrial ΔY was estimated through fluorescence changes of 5 mM safranin O as described under the Experimental section, that is, 100% correspond to 140.3 6 mV for kidney, 205.7 7.5 mV for brain, 143.4 2.8 mV for heart, and 116.0 2 mV for liver mitochondria. Conditions are as follows: MRM, mitochondria in MRM; C, mitochondria with 10 mM citrate/2 mM malate; C FC(T), mitochondria with 10 mM citrate/2 mM malate, and fluorocitrate at concentration which correspond to threshold; C FC(IC50), mitochondria with 10 mM citrate/2 mM malate, and fluorocitrate at concentration which correspond to IC50. *p 0.05 respect to C.
690 F. Scandroglio et al. effect afforded by the metabolic network will be involved in the threshold effect as previously reported [26]. Discussion
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Figure 4. Aconitase expression in different rat tissues. 30 mg mitochondrial protein from brain, heart, liver, and kidney is separated by 10% SDS–PAGE and aconitase tissue levels are analyzed by Western blot using a polyclonal anti-aconitase 2 antibody.
at 21% O2. From the tissues analyzed, a significant inactive pool (12%) of aconitase could also be found in heart (Table I), reflecting increased O2· basal formation in these tissues compared with that in liver or kidney. Also, tissues that had similar amounts of aconitase according to the Western blot and the specific activity (i.e., heart and kidney Figure 4 and Table I) presented different threshold profile curves (Figure 2C and Supplementary Figure 1 available online at http://informa healthcare.com/doi/abs/10.3109/10715762.2014.900175). These different thresholds probably reflect that for aconitase, whatever its excess of activity, the buffering
In this study, using a limited metabolic control analysis to evaluate the role of aconitase over the citric acid cycle and respiration, we showed that inhibition of aconitase could impact mitochondrial metabolism by diminishing the entry of reducing equivalents to the electron transport chain, decreasing ∆Y, and slowing the rates of O2· and H2O2 production. Extensive previous evidence from many different cells, tissues, and organisms added to the in vitro experiments’ support that mitochondrial aconitase activity is uniquely sensitive to the enhanced formation of oxygen and nitrogen reactive species in various physiological and pathological conditions [2,4,6,8,13]. In cells, active aconitase reaches a steady-state level, due to inactivation and reactivation reactions, that is related to the rates of formation and consumption of these reactive species and the levels of reductants. Moreover, due to the selectivity of O2· reactivity toward aconitase [Fe-S] cluster, aconitase activity has become a sensitive assay for measuring the variation in O2· levels inside cells and mitochondria in a
Figure 5. Redox regulation of mitochondrial aconitase. Active mitochondrial aconitase is a monomeric protein containing a [4Fe-4S] cluster which directly participates during the catalytic mechanisms promoting isocitrate formation. Reactive oxygen and nitrogen species (ROS and RNS) such as O2 ·, H2O2, ONOO-among others, react with the Fe-S cluster promoting Fe release and inactivation of the enzyme; therefore, citrate concentration will raise. [3Fe-4S]-Aconitase could be reactivated in vivo in the presence of Fe and reductants. Oxidative modification of aconitase which leads to total [Fe-S] cluster disassembly or amino acid oxidation promotes enzyme degradation. Aconitase activity is also regulated by covalent phosphorylation by a still undefined phosphorylation cascade. As kinases are activated by redox modifications, both Fe-S cluster and phosphorylation of aconitase could operate in line shifting the catalyzed reaction toward citrate formation.
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Aconitase inactivation and mitochondrial metabolic control 691
wide variety of cell types and conditions [4,6,8,13]. However, the impact of deliberate aconitase inhibition over mitochondrial metabolism and ROS production has not been explored until this work. Herein, we found that heart and brain aconitase exhibited the highest flux control coefficient values and these organs also shown that only 5% aconitase inhibition is needed to impact mitochondrial respiration (threshold values, ~95–98%). Interestingly, the tissue differences shown herein support tissue specificity observed in patients with Friedreich ataxia in which while frataxin depletion is found in all tissues and Fe-S cluster protein-deficiency are spread out, symptoms reflect aconitase deficiency in brain and heart (reviewed in [28]). To operate as a redox sensor, mitochondrial aconitase has to be reversibly inactivated in vivo, as effectively occurs. For instance, during cardiac ischemia/reperfusion in rats, aconitase exhibited a loss and a regain in activity [29]. Aconitase inhibition under short reperfusion not only would limit O2· and H2O2 production by electron transport chain as we showed herein for heart mitochondria, but also prevents Ca2 overload by reducing the proton gradient across inner mitochondrial membrane and could operate as part of preconditioning beneficial effects. Mitochondrial antioxidant defenses (i.e., MnSOD, glutathione peroxidase, or peroxiredoxin III) mitigate damage produced by ROS or ·NO-derived species but a different level of control is given by mechanisms that diminish mitochondrial electrochemical gradient and thereby O2· formation (i.e., uncoupling proteins, K-ATP channels) or substrates supply to electron transport chain. In this work, we showed that inhibition of mitochondrial aconitase could operate as a rapid switch to slow O2· and H2O2 production by electron transport chain particularly in brain and heart where a significant mitochondrial depolarization was observed. In addition, and as a consequence of redox post-translational modification of mitochondrial proteins, ROS can either attenuate or promote the generation of more ROS. In this regard, in vitro experiments of VasquezVivar and coworkers [30] support that reaction of O2· with aconitase yield H2O2 and Fe2 which led to hydroxyl radical (·OH) formation by a Fenton mechanism; the authors envisioned inactivation of aconitase as a source of free iron and free radical damage. Overexpression of mitochondrial aconitase in rat mesencephalic cell cultures by adenoviral vectors led to an increased paraquat sensitivity to these cells. This effect was translated by an augmented cell death accompanying higher levels of H2O2 and Fe2 [31]. Also, mitochondrial aconitase knockdown attenuated paraquat-induced dopaminergic cell death [32], but these cells exhibited considerable lower basal and stimulated oxygen consumption rates showing that an overall decrease in mitochondrial metabolism are also implicated in protection mechanism. In summary, while it seems clear that aconitase is a source of iron due to O2·-mediated inactivation, the relevance of this source in oxidant-mediated damage can be questioned. That is because under physiological conditions, where natural mitochondrial iron chelators are present (i.e., citrate, glutamylcitrate, and
ADP), iron protein chaperones (i.e., frataxin) are active, and H2O2 would be quickly consumed by GSH peroxidase or peroxiredoxin III, and these mechanisms could be envisioned to trigger irreversible mitochondrial damage. Modulation of the citric acid cycle and mitochondrial ROS production by aconitase can operate as part of a coordinated mitochondrial redox regulation. For instance, using a proteomic technique, mitochondrial thiol proteins which reacted with low (physiologically relevant) levels of H2O2 were identified and postulated to afford some kind of redox regulation in mitochondrial metabolism [33]. Among them, pyruvate dehydrogenase kinase 2, a major regulator of pyruvate dehydrogenase complex, was revealed to be very sensitive by oxidation of Cys residues 45 and 392 which result in its inactivation, thereby decreasing pyruvate dehydrogenase complex phosphorylation and subsequently increasing acetyl CoA formation [34], potentially favoring ROS generation. Interestingly, the same ROS levels inactivated aconitase through Fe-S cluster oxidation; therefore, citric acid would be slowed, limiting the increase in supply of substrate to electron transport chain, supporting the concept that mitochondrial ROS can modulate several sites of mitochondrial metabolism in a coordinate manner. This mechanism, in which increased formation of acetyl CoA is formed but aconitase is inhibited, might shift the balance between carbohydrate and fat metabolism and help to explain the purpose of maintaining an extremely O2·–sensitive aconitase during evolution [14]. In addition, supporting data from homozygous MnSOD knock-out mice which die within a week of birth and display significantly decreased aconitase activity had significant cardiomyopathy with accumulation of fat in liver and muscle [35]. Also, heterozygous MnSOD knockout mice are insulin-resistant [36] and mitochondriatargeted antioxidants lower fat content, and protect against insulin resistance [37], supporting the concept that controlled O2· production may regulate the flux into the electron transport chain for oxidative phosphorylation or may channel citrate into the synthesis of fats through its effect on aconitase. Besides redox regulation of the Fe-S cluster of aconitase, post-translational modifications of this enzyme have also been found. In particular, phosphorylated aconitase was found in skeletal muscle of human [38] and in hearts from type 1 diabetic rats [39]. Exercising accompanied aconitase dephosphorylation and augmented catalytic activity toward isocitrate formation [38]. Augmented phosphorylation of mitochondrial aconitase mediated by protein kinase C in diabetic rat hearts was found to be associated with an increase in its reverse activity, that is, toward citrate formation [39]. As citrate concentration is increased in the heart of both acute and chronic diabetes [40], this additional mechanism to redox modification could operate together to stop citric acid cycle leading to accumulation of upstream metabolites (Figure 5). In addition, as H2O2 can modulate phosphorylation cascades, both mechanisms could operate in a coordinated fashion as suggested in experiments using intact mitochondria exposed to H2O2 [41]. Additionally, other
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692 F. Scandroglio et al. post-translational modifications related to nitro-oxidative stress have been detected in mitochondrial aconitase, either in vitro (glutathionylation) or in vivo (s-nitrosylation) [42,43]. Although its biological significance is still unclear, it is certain that mitochondrial aconitase is plausible of multiple redox activity-tuning. Metabolism of carbohydrates, fats, and proteins merge at the citric acid cycle, which then directly generates ATP and provides NADH as well as FADH2 as substrates for ATP production through the electron transport chain. A major mechanism for the regulation of cellular energy metabolism is achieved through the citric acid cycle enzymes, citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase, which are classically considered as under negative allosteric inhibition by their products, abundant ATP, and a high ratio of NADH/NAD in mammalian cells. In particular, a-ketoglutarate dehydrogenase which seems to be the master regulator over the citric acid cycle is also a target and a generator of ROS [44], but its sensitivity to ROS-mediated inactivation is significantly lower than aconitase. As stated in [44], inactivation of aconitase can be eluded by other substrates to the citric acid cycle, that is, by providing a-ketoglutarate from glutamate transamination instead of pyruvate so that NADH supply to electron transport chain would be maintained. Herein, using a limited metabolic control approach, we provide evidence that aconitase can subtly regulate the flux of metabolites though the citric acid cycle and therefore modulate the flux of electron transport chain and ROS production by mitochondria. The regulatory role of aconitase seems to have tissue (or cell) specificity, and the control of metabolic flux afforded by aconitase can be achieved by at least two mechanisms: a) redox regulation of the Fe-S cluster and b) phosphorylation/dephosphorylation (Figure 5). Under conditions that promote 3Fe-4S aconitase and/or phosphorylated enzyme, citrate concentration would rise, the rate of the citric acid cycle would be slowed and free radical production by the electron transport chain will be rapidly diminished. Acknowledgments We thank Dr. Celia Quijano, Universidad de la República, Uruguay, to her helpful comments, and Dr. Frederick E. Domann, University of Iowa, USA, for careful reading and English editing. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grants from the Agencia Nacional de Innovación e Investigación (ANII, FCE_ 398 to LC and FCE_2486 to RR) and Comisión Sectorial de Investigación Científica, Universidad de la República.
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Supplementary material available online Supplementary Figure 1.
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ORIGINAL ARTICLE
Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production F. Scandroglio, V. Tórtora, R. Radi & L. Castro
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Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay Abstract The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC50 for aconitase inhibition ranging from 7 to 24 mM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient ( 0.95). Aconitase-specific activity varied widely among tissues examined from ~60 mU/mg in liver to 321 mU/mg in kidney at 21% O2. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O2 reflecting aconitase inactivation by oxygen-derived oxidants at 21% O2. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O2· and H2O2 formation by the respiratory chain. Keywords: aconitase 2, free radicals, hydrogen peroxide, metabolic control, mitochondria, superoxide radical Abbreviations: O2·, Superoxide radical; ·NO, nitric oxide; ONOO, peroxynitrite; CO3·, carbonate radical; H2O2, hydrogen peroxide; MRM, mitochondrial respiration medium; RCR, respiratory control ratio
Introduction Mitochondrial aconitase, an enzyme that catalyzes the reversible isomerization of citrate to isocitrate via cisaconitate in the citric acid cycle, contains an [4Fe-4S] prosthetic group in which one of the irons, Fea, is not ligated to a protein residue, and thus can bind to hydroxyl groups of substrates or water. Superoxide radical (O2·) and reactive species derived from its reaction with nitric oxide, ·NO (i.e., peroxynitrite, ONOO and carbonate radical, CO3·), rapidly react with the Fe-S cluster, yielding an inactive protein in vitro [1–6] that can be reactivated by reductants (such as glutathione) and iron in vivo. Nitric oxide also interacts directly and reversible with aconitase [1]; however, sustained ·NO slowly promotes total Fe-S cluster disassembly [3,7]. Proteomic analysis of mitochondria from animal models of sepsis, diabetes, and aging revealed aconitase inactivation and nitration in vivo [8–12], showing that mitochondrial aconitase is a main target of reactive species in vivo. Due to the specificity of O2· reaction toward [4Fe-4S] aconitase cluster, the relationship between inactive and active aconitase has been used as a method to measure steady-state O2· concentration in mitochondria [4,13].
Mitochondrial aconitase evolved from its a-proteobacterial ancestor aconitase B which is more sensitive to O2· than aconitase A that originated the cytosolic counterpart (cytosolic aconitase, also known as iron-responsive protein 1). The presence of an exquisitely O2·–sensitive enzyme within the mitochondrial matrix led to the speculation that mitochondrial aconitase may provide a redox control mechanism of the citric acid cycle and suggested that the electron transport chain could somehow control O2· production by mitochondria [14,15]. Though indirect evidence supports this idea [15], the role of aconitase as a redox rheostat has not been proved yet. Flux control theory determines the control that various steps in a pathway have over the global flux of that pathway [16]. Based on this theory, metabolic control analysis has been applied to the study of mitochondrial oxidative phosphorylation in order to explain and support tissue specificity in human mitochondrial diseases and the evidence that a reduction in the activities of some respiratory chain complexes is associated with aging or neurodegenerative diseases [16–19]. Mitochondrial respiratory complex proteins are present in excess of the amount required to meet normal metabolic demand; therefore, submaximal inhibition of the enzyme does not
Correspondence: Laura Castro, Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Avenida General Flores 2125, 11800 Montevideo, Uruguay. Tel: (598)-2924-3414 ext 3402. Fax: (598)-2924-9563. E-mail: [email protected] (Received date: 15 January 2014; Accepted date: 27 February 2014; Published online: 28 March 2014)
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affect the overall metabolic pathway until a threshold level of inhibition is reached, depending somehow on the control exerted that such enzyme has on the whole pathway. Also, metabolic control analysis afforded by citric acid enzymes over respiration has been used to characterize the impact of mitochondrial bioenergetics in dopaminergic cells [20]. Herein, using isolated mitochondria from different rat tissues with varied metabolic profiles, we determined the flux control coefficient elicited by aconitase over the citric acid cycle and the respiratory chain. We aimed to determine the threshold level of aconitase inhibition that produces metabolic impact and how aconitase inhibition impacts mitochondrial oxidant production, that is, O2· and hydrogen peroxide (H2O2). Metabolic control analysis was performed in order to assess the role of aconitase as a redox sensor to test the hypothesis that increased levels of mitochondrial oxidants will impact on aconitase activity, thereby diminishing the reducing equivalents entering into the electron transport chain thus stopping the propagation of free radical generation by mitochondria. Experimental Reagents Chemicals were supplied by Sigma Chemical Co. (St. Louis, MO, USA) or Applichem GmbH (Germany). Amplex red was obtained from Molecular Probes-Invitrogen (Eugene, OR, USA). Mitochondrial isolation Intact liver, kidney, hearts, and brain mitochondria were prepared by differential centrifugation as described previously [21] from adult Wistar rats (200–250 g). Liver mitochondria were isolated from overnight fasted animals. To obtain heart mitochondria, hearts were minced into small pieces with a tissue grinder before homogenization with the Potter-Elvehjem. Due to the purification procedure, brain mitochondria obtained in this study consist preferentially of non-synaptosomal mitochondria. All experiments were performed with the approval of the Institutional Animal Welfare Committee. Mitochondrial pellets were resuspended in a minimal volume of MRM buffer (0.3 M sucrose, 5 mM potassium phosphate, 5 mM morpholinepropanesulfonic acid, 1 mM EGTA, 0.1% bovine serum albumin, pH 7.4), and protein concentration was determined according to the Bradford method. In some experiments, mitochondria were exposed to three cycles of freeze–thaw in order to obtain broken mitochondria. Aconitase activity Aconitase activity was measured in fresh sonicated mitochondria (15 mg/ml) with the addition of citrate and followed by the appearance of cis-aconitate at 240 nm in 100 mM Tris–HCl of pH 7.4. Inhibitor titration using
increasing concentrations of fluorocitrate (0–60 mM) was performed. The concentration of fluorocitrate needed to inactivate 50% of aconitase activity (IC50) was calculated by adjusting the plot to a single exponential function. Maximal aconitase-specific activity found for the different tissues was determined after the incubation of fresh sonicated mitochondria for 30 min under very low oxygen tension ( 2% oxygen) in a Coy lab hypoxic chamber. Oxygen consumption measurements Oxygen consumption elicited by substrates was evaluated in fresh mitochondrial (0.01–0.05 mg/ml) preparations by high-resolution respirometry (Oroboros®, Insbruck, Austria) at 37°C, in MRM buffer. Calibration with airsaturated buffer was performed daily. Data were collected and analyzed using software DatLab (Oroboros) displaying real-time oxygen concentration and oxygen flux, that is, the negative-time derivative course of oxygen concentration. Complex I and Complex II-dependent respiration was routinely assessed using 10 mM glutamate/2 mM malate or 10 mM succinate. Respiratory control ratio was evaluated after the addition of 1 mM ADP, and residual oxygen consumption was determined by the addition of 5 mM antimycin-A. As citrate alone did not support respiration in our mitochondrial preparations, we evaluated aconitase through citric acid cycle-complex I-dependent respiration by adding 10 mM citrate/2 mM malate as substrates as previously reported [20]. In the latter conditions, titrations with fluorocitrate were performed during state 3 respiration. Aconitase-driven respiration was confirmed by recovered respiration afforded by 5 mM isocitrate after fluorocitrate titrations. Aconitase expression in different rat tissues Mitochondrial proteins (30 mg) from brain, heart, liver, and kidney were separated by 10% SDS–PAGE, and aconitase tissue levels were analyzed by Western blot using a polyclonal anti-aconitase 2 antibody from ABCAM. Immunoreactive proteins were detected and quantified using the Odissey Infrared Imaging System. Mitochondrial hydrogen peroxide production and membrane potential determination Mitochondrial H2O2 release arising from O2· dismutation [22] was measured in 0.1 mg protein/ml intact mitochondrial suspensions in MRM buffer at 37°C, with continuous stirring. Amplex Red (25 mM) oxidation was followed in the presence of 0.5 U/ml horseradish peroxidase and 10 mM citrate/2 mM malate in the presence of 1 mM ADP. In some conditions, fluorocitrate was added at two different concentrations: equal to threshold value and equal to IC50. Amplex Red is oxidized, generating resorufin, which can be detected fluorimetrically using a plate reader fluorescence spectrophotometer (Fluostar) lex 530 nm, lem 590 nm. Controls conducted in the absence of
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686 F. Scandroglio et al. mitochondria or in the absence of peroxidase indicate that nonspecific probe oxidation is negligible as reported previously [22]. As a positive control, antimycin A in the presence of substrates that triggers O2· formation by Complex III was used and resulted in 6–10 times higher H2O2 formation than control values. Calibration was performed by adding H2O2 at known concentrations (e240nm 43.6 M–1 cm–1). Mitochondrial ∆Y was estimated through fluorescence changes of 5 mM safranin O at lex 486 nm and lem 585 nm at 37°C, as described in [22,23]. For these purposes, mitochondria were isolated in potassium-free medium as described in [23]. Incubation conditions were the same as for H2O2 release measurements. Data obtained were calibrated using a K gradient. The ∆Y value obtained for each K concentration was determined using the Nernst equation, assuming intramitochondrial [K] to be 120 mM, and plotted against measured fluorescence values to generate a calibration curve for each tissue. Calculation of flux control coefficients and respiratory threshold Flux control coefficients were calculated according to the metabolic control theory [16]. Control coefficients quantitatively describe the control exerted by and enzyme in a metabolic step or network over substrate flux. The flux control coefficient can be defined as the fractional change in pathway flux of a metabolic network under steady-state conditions, induced by a fractional change in the individual step under consideration. A consequence of the definition of control coefficient is the summation theorem, stating that in a metabolic network, the sum of the flux control coefficient of each step of the network is equal to one. In this case, we are analyzing only one step (aconitase) of the entire pathway. For aconitase-dependent oxidative flux (respiration) in mitochondria:
dJO 2 d (Inhibitor) I CJ dVc d (Inhibitor)
(1)
where CIJ is the flux control coefficient of aconitase, dVc/d(Inhibitor) is the rate of change of aconitase activity (individual step), and dJO2/d(Inhibitor) is the rate of change of respiration (global flux), at low concentrations of the complex inhibitor. Fluorocitrate, a suicide aconitase inhibitor [24], was used to performed aconitase activity and aconitase-dependent respiration titrations in order to assess control coefficients and threshold values according to [20,25]. The threshold curves come from the titration curves. Each point of a threshold curve represents the respiratory rate inhibition percentage as a function of inhibition percentage of aconitase activity for the same fluorocitrate concentration.
General procedures All experiments reported herein were reproduced at least thrice, and results shown correspond to one representative experiment of each one or the mean SD. For statistical analysis, independent t-tests were performed, with a 0.05, P 0.05. Graphics and mathematical fits to experimental data were performed using OriginPro 8 (OriginLab Corporation). Results Fluorocitrate titration of aconitase in different rat tissues In order to perform a metabolic control analysis, the measurement of the impact of increasing concentration of specific inhibitors on enzyme activities versus substrate specific respiration is used to obtain titration curves for graphical determination of flux control coefficient, an index of enzyme contribution to flux control in the entire pathway. Therefore, it is necessary to evaluate aconitase enzyme activity as well as aconitase-driven NADH production and oxygen consumption. Aconitase catalytic activity and aconitase-dependent respiration were titrated with the specific inhibitor fluorocitrate both as isolated reaction step in sonicated mitochondria and as respiratory chain integrated steps in intact mitochondria, respectively. In this way, it was possible to determine at each concentration of fluorocitrate used, the percentage of inhibition of aconitase activity in permeabilized mitochondria and how this inhibition affects the respiratory flux. Figure 1 shows that increasing concentrations of fluorocitrate (0–40 mM) decreased aconitase-catalytic activity (A) and citratedependent respiration in kidney mitochondria (B). In respirometry experiments, after successive fluorocitrate additions, isocitrate addition resulted in recovery of oxygen consumption showing the specificity of aconitasedependent respiration in this assay (Figure 1B) and the integrity of the other steps of the citric acid cycle and electron transport chain. Possible effects of fluorocitrate in electron transport chain were also excluded by performing experiments with broken mitochondria in which NADH-dependent oxygen consumption rates were evaluated with or without fluorocitrate which showed similar rates (not shown). Similar plots were obtained for other tissues analyzed (heart, brain, and liver—not shown). The respiratory control ratio (RCR) calculated as the ratio of oxygen consumption rates after (state 3) and before (state 4) ADP addition using citrate as substrate was always lower than for glutamate/malate (RCR from 5 to 7) or succinate (RCR for from 3 to 5), probably reflecting citrate constraints to reach the mitochondrial matrix. The flux control coefficient (CIJ) for aconitase in the different organs analyzed was calculated as described earlier and gave the CIJ 0.54 for aconitase in kidney. The threshold level of aconitase inhibition over respiration was
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Figure 1. Fluorocitrate titrations of aconitase. (A) Aliquots of fresh sonicated kidney mitochondria are incubated with 100 mM citrate in 100 mM Tris–HCl, pH 7.4, in the absence or in the presence of increasing mM concentration of fluorocitrate as indicated in the right of panel A. Aconitase activity is measured following cis-aconitase formation at 240 nm. (B) A typical oxygen consumption trace for fresh kidney mitochondria preparations. Measurements are performed at 37°C in the 2-ml chamber of the Oroboros Oxygraph-2k in mitochondrial respiration medium. The following are then added: Mt, 0.01 mg/ml mitochondria; Malate/citrate, 10 mM citrate/2 mM malate; 1 mM ADP; 30 mM and 50 mM Fluorocitrate; 5 mM Isocitrate. Blue line: oxygen concentration (mM); red line: oxygen flow (pmol/sec.mg). Color available at the web version of this article.
determined from a curve that expressed the rate of oxygen consumption as a function of aconitase inhibition (Figure 2C) which came from combining y-axes of A and B plots in Figure 2. Kidney aconitase inhibition threshold was found to be 63%, beyond which respiration decreased rapidly. Supplementary Figure 1 available online at http:// informahealthcare.com/doi/abs/10.3109/10715762.2014. 900175 shows data obtained for brain, heart, and liver mitochondria. According to the threshold curves from the different tissues analyzed, two distinct types of behavior can be distinguished as previously reported [26]. For liver and kidney, a plateau is observed followed by a stepped breakage, whereas for the heart and brain threshold curves the plateau is not evident (Figure 2C and Supplementary Figure 1 available online at http://informahealthcare.com/ doi/abs/10.3109/10715762.2014.900175). Table I summarizes the data obtained for the different organs analyzed. Titrations with fluorocitrate showed tissue variations with IC50 of 24, 24, 7, and 12 mM in kidney, liver, brain, and heart, respectively (Table I). Brain tissue exhibited the highest flux control coefficient (0.98). Before significant changes in oxygen consump-
tion were observed, aconitase inhibition thresholds were determined to be 63, 69, 95, and 98% for kidney, liver, brain, and heart, respectively (Table I), meaning that in liver and kidney, ~40% inhibition of aconitase is required to slow respiration, but less than 5% inhibition of aconitase will impact on brain and 2% in heart mitochondrial respiration. Although aconitase is not considered to be a rate-limiting enzyme in the citric acid cycle, its inhibition could impact the generation of NADH by the citric acid cycle and its supply to electron transport chain as could be envisaged by flux control coefficient obtained, especially for brain and heart. Membrane potential and hydrogen peroxide production in aconitase-inhibited mitochondria As mitochondrial O2· and H2O2 production increases when there is abundance of reduced transport chain complexes which led to mitochondrial inner membrane hyperpolarization [27], we measured mitochondrial membrane potential in control and aconitase-inhibited mitochondria. As shown in Figure 3, H2O2 released by
688 F. Scandroglio et al. Table I. Comparative parameters obtained from rat tissues. SA (nmol/mg.ml) Tissue IC50 (mM) Kidney Liver Brain Heart
24 24 7 12
CIJ 0.54 0.018 0.13 0.008 0.98 0.023 0.80 0.015
Threshold 21% O2 63 69 95 98
2% O2
321 25 307 30 59.2 3 55 7 115 15 163 12* 296 13 334 14*
A, specific activity; CJI, flux control coefficient; IC50, fluorocitrate S concentration needed to inhibit aconitase activity by 50%; Threshold, level of aconitase inhibition needed to significantly affect respiration. *p 0.05 significantly to 21% O2.
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mitochondria from all tissues analyzed increased when citrate was present as substrate and an accompanying increase in ∆Y across inner mitochondrial membrane (compare first and second bars in graphs from left and right columns in Figure 3). Hydrogen peroxide generation rate varied significantly among the studied tissue. In the presence of citrate and ADP, H2O2 production rate was 10.6 0.8 nmol.min 1.mg 1 for kidney, 1.8 0.2 nmol.min 1.mg 1 for brain, 7.3 0.3 nmol. min 1.mg 1 for heart, and 15 0.3 nmol.min 1.mg 1 for liver mitochondria. These differences were in line with tissue variations reported for other substrates [22]. When fluorocitrate was added at a concentration which correlates to that obtained at threshold level when respiration is compromised, released H2O2 is decreased, and a drop in ∆Y was observed (Figure 3E–H). The effects of fluorocitrate were higher at the concentration that correlates with the obtained IC50 for each tissue. Interestingly, these effects were statistically significant in brain and heart, the tissues with the highest CIJ. Aconitase expression and maximal aconitase activity in different rat tissues
Figure 2. Measurement of mitochondrial respiratory threshold and calculation of flux control coefficient elicited by aconitase. (A) Fresh kidney mitochondria (15 mg/ml) are sonicated, and aconitase activity is immediately assayed using citrate as substrate and increasing concentrations of fluorocitrate (0–60 m M). Aconitase activity is expressed as percentage of the control. The concentration of fluorocitrate needed to inactivate 50% of aconitase activity (IC50) is calculated by adjusting the plot to a single exponential function. (B) Oxygen consumption in the presence of fluorocitrate is determined when the flux reached a steady state after each addition. Oxygen consumption (respiration) is expressed as percentage of citrate-dependent respiration in the presence of ADP (State 3). (C) Threshold curves from fluorocitrate titration are constructed by combining y-axis of the graphs A and B. Metabolic
Aconitase-specific activity varied significantly over the tissues studied herein (Table I). Mitochondrial aconitase is a constitutive protein, but the amount of the enzyme differed in the organs analyzed (Figure 4). As observed in the Western blot, though aconitase expression in brain and kidney was similar, specific activity was not. As it was previously reported, a fraction of inactive aconitase is found in basal condition which reflects O2· steadystate production in cells [4]. Therefore, we explored if the discrepancies observed between specific activity and expression, particularly for brain aconitase could be attributed to a pool of inactive aconitase present in this tissue. Incubation of sonicated mitochondria in a very low O2 atmosphere ( 2% O2) for 30 min yielded 42% higher brain aconitase-specific activity than that observed
control analysis is used to determine the flux control coefficient for aconitase considering the rate of change of oxygen consumption (entire flux) at low concentration of fluorocitrate and the rate of change of activity as explained in the text.
Aconitase inactivation and mitochondrial metabolic control 689
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Figure 3. Aconitase inhibition leads to a decrease in mitochondrial membrane potential and a diminished hydrogen peroxide production. (A–D) Hydrogen peroxide fluxes were measured in 0.1 mg/ml mitochondrial suspensions following the oxidation of Amplex red as described under the Experimental section. 100% correspond to: 10.6 0.8 nmol/mg.min for kidney, 1.8 0.2 nmol/mg.min for brain, 7.3 0.3 nmol/mg.min for heart, and 15 0.3 nmol/mg.min for liver mitochondria. (E–H) Mitochondrial ΔY was estimated through fluorescence changes of 5 mM safranin O as described under the Experimental section, that is, 100% correspond to 140.3 6 mV for kidney, 205.7 7.5 mV for brain, 143.4 2.8 mV for heart, and 116.0 2 mV for liver mitochondria. Conditions are as follows: MRM, mitochondria in MRM; C, mitochondria with 10 mM citrate/2 mM malate; C FC(T), mitochondria with 10 mM citrate/2 mM malate, and fluorocitrate at concentration which correspond to threshold; C FC(IC50), mitochondria with 10 mM citrate/2 mM malate, and fluorocitrate at concentration which correspond to IC50. *p 0.05 respect to C.
690 F. Scandroglio et al. effect afforded by the metabolic network will be involved in the threshold effect as previously reported [26]. Discussion
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Figure 4. Aconitase expression in different rat tissues. 30 mg mitochondrial protein from brain, heart, liver, and kidney is separated by 10% SDS–PAGE and aconitase tissue levels are analyzed by Western blot using a polyclonal anti-aconitase 2 antibody.
at 21% O2. From the tissues analyzed, a significant inactive pool (12%) of aconitase could also be found in heart (Table I), reflecting increased O2· basal formation in these tissues compared with that in liver or kidney. Also, tissues that had similar amounts of aconitase according to the Western blot and the specific activity (i.e., heart and kidney Figure 4 and Table I) presented different threshold profile curves (Figure 2C and Supplementary Figure 1 available online at http://informa healthcare.com/doi/abs/10.3109/10715762.2014.900175). These different thresholds probably reflect that for aconitase, whatever its excess of activity, the buffering
In this study, using a limited metabolic control analysis to evaluate the role of aconitase over the citric acid cycle and respiration, we showed that inhibition of aconitase could impact mitochondrial metabolism by diminishing the entry of reducing equivalents to the electron transport chain, decreasing ∆Y, and slowing the rates of O2· and H2O2 production. Extensive previous evidence from many different cells, tissues, and organisms added to the in vitro experiments’ support that mitochondrial aconitase activity is uniquely sensitive to the enhanced formation of oxygen and nitrogen reactive species in various physiological and pathological conditions [2,4,6,8,13]. In cells, active aconitase reaches a steady-state level, due to inactivation and reactivation reactions, that is related to the rates of formation and consumption of these reactive species and the levels of reductants. Moreover, due to the selectivity of O2· reactivity toward aconitase [Fe-S] cluster, aconitase activity has become a sensitive assay for measuring the variation in O2· levels inside cells and mitochondria in a
Figure 5. Redox regulation of mitochondrial aconitase. Active mitochondrial aconitase is a monomeric protein containing a [4Fe-4S] cluster which directly participates during the catalytic mechanisms promoting isocitrate formation. Reactive oxygen and nitrogen species (ROS and RNS) such as O2 ·, H2O2, ONOO-among others, react with the Fe-S cluster promoting Fe release and inactivation of the enzyme; therefore, citrate concentration will raise. [3Fe-4S]-Aconitase could be reactivated in vivo in the presence of Fe and reductants. Oxidative modification of aconitase which leads to total [Fe-S] cluster disassembly or amino acid oxidation promotes enzyme degradation. Aconitase activity is also regulated by covalent phosphorylation by a still undefined phosphorylation cascade. As kinases are activated by redox modifications, both Fe-S cluster and phosphorylation of aconitase could operate in line shifting the catalyzed reaction toward citrate formation.
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Aconitase inactivation and mitochondrial metabolic control 691
wide variety of cell types and conditions [4,6,8,13]. However, the impact of deliberate aconitase inhibition over mitochondrial metabolism and ROS production has not been explored until this work. Herein, we found that heart and brain aconitase exhibited the highest flux control coefficient values and these organs also shown that only 5% aconitase inhibition is needed to impact mitochondrial respiration (threshold values, ~95–98%). Interestingly, the tissue differences shown herein support tissue specificity observed in patients with Friedreich ataxia in which while frataxin depletion is found in all tissues and Fe-S cluster protein-deficiency are spread out, symptoms reflect aconitase deficiency in brain and heart (reviewed in [28]). To operate as a redox sensor, mitochondrial aconitase has to be reversibly inactivated in vivo, as effectively occurs. For instance, during cardiac ischemia/reperfusion in rats, aconitase exhibited a loss and a regain in activity [29]. Aconitase inhibition under short reperfusion not only would limit O2· and H2O2 production by electron transport chain as we showed herein for heart mitochondria, but also prevents Ca2 overload by reducing the proton gradient across inner mitochondrial membrane and could operate as part of preconditioning beneficial effects. Mitochondrial antioxidant defenses (i.e., MnSOD, glutathione peroxidase, or peroxiredoxin III) mitigate damage produced by ROS or ·NO-derived species but a different level of control is given by mechanisms that diminish mitochondrial electrochemical gradient and thereby O2· formation (i.e., uncoupling proteins, K-ATP channels) or substrates supply to electron transport chain. In this work, we showed that inhibition of mitochondrial aconitase could operate as a rapid switch to slow O2· and H2O2 production by electron transport chain particularly in brain and heart where a significant mitochondrial depolarization was observed. In addition, and as a consequence of redox post-translational modification of mitochondrial proteins, ROS can either attenuate or promote the generation of more ROS. In this regard, in vitro experiments of VasquezVivar and coworkers [30] support that reaction of O2· with aconitase yield H2O2 and Fe2 which led to hydroxyl radical (·OH) formation by a Fenton mechanism; the authors envisioned inactivation of aconitase as a source of free iron and free radical damage. Overexpression of mitochondrial aconitase in rat mesencephalic cell cultures by adenoviral vectors led to an increased paraquat sensitivity to these cells. This effect was translated by an augmented cell death accompanying higher levels of H2O2 and Fe2 [31]. Also, mitochondrial aconitase knockdown attenuated paraquat-induced dopaminergic cell death [32], but these cells exhibited considerable lower basal and stimulated oxygen consumption rates showing that an overall decrease in mitochondrial metabolism are also implicated in protection mechanism. In summary, while it seems clear that aconitase is a source of iron due to O2·-mediated inactivation, the relevance of this source in oxidant-mediated damage can be questioned. That is because under physiological conditions, where natural mitochondrial iron chelators are present (i.e., citrate, glutamylcitrate, and
ADP), iron protein chaperones (i.e., frataxin) are active, and H2O2 would be quickly consumed by GSH peroxidase or peroxiredoxin III, and these mechanisms could be envisioned to trigger irreversible mitochondrial damage. Modulation of the citric acid cycle and mitochondrial ROS production by aconitase can operate as part of a coordinated mitochondrial redox regulation. For instance, using a proteomic technique, mitochondrial thiol proteins which reacted with low (physiologically relevant) levels of H2O2 were identified and postulated to afford some kind of redox regulation in mitochondrial metabolism [33]. Among them, pyruvate dehydrogenase kinase 2, a major regulator of pyruvate dehydrogenase complex, was revealed to be very sensitive by oxidation of Cys residues 45 and 392 which result in its inactivation, thereby decreasing pyruvate dehydrogenase complex phosphorylation and subsequently increasing acetyl CoA formation [34], potentially favoring ROS generation. Interestingly, the same ROS levels inactivated aconitase through Fe-S cluster oxidation; therefore, citric acid would be slowed, limiting the increase in supply of substrate to electron transport chain, supporting the concept that mitochondrial ROS can modulate several sites of mitochondrial metabolism in a coordinate manner. This mechanism, in which increased formation of acetyl CoA is formed but aconitase is inhibited, might shift the balance between carbohydrate and fat metabolism and help to explain the purpose of maintaining an extremely O2·–sensitive aconitase during evolution [14]. In addition, supporting data from homozygous MnSOD knock-out mice which die within a week of birth and display significantly decreased aconitase activity had significant cardiomyopathy with accumulation of fat in liver and muscle [35]. Also, heterozygous MnSOD knockout mice are insulin-resistant [36] and mitochondriatargeted antioxidants lower fat content, and protect against insulin resistance [37], supporting the concept that controlled O2· production may regulate the flux into the electron transport chain for oxidative phosphorylation or may channel citrate into the synthesis of fats through its effect on aconitase. Besides redox regulation of the Fe-S cluster of aconitase, post-translational modifications of this enzyme have also been found. In particular, phosphorylated aconitase was found in skeletal muscle of human [38] and in hearts from type 1 diabetic rats [39]. Exercising accompanied aconitase dephosphorylation and augmented catalytic activity toward isocitrate formation [38]. Augmented phosphorylation of mitochondrial aconitase mediated by protein kinase C in diabetic rat hearts was found to be associated with an increase in its reverse activity, that is, toward citrate formation [39]. As citrate concentration is increased in the heart of both acute and chronic diabetes [40], this additional mechanism to redox modification could operate together to stop citric acid cycle leading to accumulation of upstream metabolites (Figure 5). In addition, as H2O2 can modulate phosphorylation cascades, both mechanisms could operate in a coordinated fashion as suggested in experiments using intact mitochondria exposed to H2O2 [41]. Additionally, other
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692 F. Scandroglio et al. post-translational modifications related to nitro-oxidative stress have been detected in mitochondrial aconitase, either in vitro (glutathionylation) or in vivo (s-nitrosylation) [42,43]. Although its biological significance is still unclear, it is certain that mitochondrial aconitase is plausible of multiple redox activity-tuning. Metabolism of carbohydrates, fats, and proteins merge at the citric acid cycle, which then directly generates ATP and provides NADH as well as FADH2 as substrates for ATP production through the electron transport chain. A major mechanism for the regulation of cellular energy metabolism is achieved through the citric acid cycle enzymes, citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase, which are classically considered as under negative allosteric inhibition by their products, abundant ATP, and a high ratio of NADH/NAD in mammalian cells. In particular, a-ketoglutarate dehydrogenase which seems to be the master regulator over the citric acid cycle is also a target and a generator of ROS [44], but its sensitivity to ROS-mediated inactivation is significantly lower than aconitase. As stated in [44], inactivation of aconitase can be eluded by other substrates to the citric acid cycle, that is, by providing a-ketoglutarate from glutamate transamination instead of pyruvate so that NADH supply to electron transport chain would be maintained. Herein, using a limited metabolic control approach, we provide evidence that aconitase can subtly regulate the flux of metabolites though the citric acid cycle and therefore modulate the flux of electron transport chain and ROS production by mitochondria. The regulatory role of aconitase seems to have tissue (or cell) specificity, and the control of metabolic flux afforded by aconitase can be achieved by at least two mechanisms: a) redox regulation of the Fe-S cluster and b) phosphorylation/dephosphorylation (Figure 5). Under conditions that promote 3Fe-4S aconitase and/or phosphorylated enzyme, citrate concentration would rise, the rate of the citric acid cycle would be slowed and free radical production by the electron transport chain will be rapidly diminished. Acknowledgments We thank Dr. Celia Quijano, Universidad de la República, Uruguay, to her helpful comments, and Dr. Frederick E. Domann, University of Iowa, USA, for careful reading and English editing. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grants from the Agencia Nacional de Innovación e Investigación (ANII, FCE_ 398 to LC and FCE_2486 to RR) and Comisión Sectorial de Investigación Científica, Universidad de la República.
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Supplementary material available online Supplementary Figure 1.
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