Tài liệu Báo cáo Y học: Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species pptx

Eur J Biochem 269, 5004–5015 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03204.x Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species Victoria I Bunik1 and Christian Sievers2 A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia; 2Physiological Chemistry Institute of Eberhard-Karls-University, Tuebingen, Germany Self-regulation of the 2-oxo acid dehydrogenase complexes during catalysis was studied Radical species as side products of catalysis were detected by spin trapping, lucigenin fluorescence and ferricytochrome c reduction Studies of the complexes after converting the bound lipoate or FAD cofactors to nonfunctional derivatives indicated that radicals are generated via FAD In the presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide anion radical was detected In the absence of oxygen, a proteinbound radical concluded to be the thiyl radical of the complex-bound dihydrolipoate was trapped by a-phenylN-tert-butylnitrone Another, carbon-centered, radical was trapped in anaerobic reaction of the complex with 2-oxoglutarate and CoA by 5,5¢-dimethyl-1-pyrroline-N-oxide (DMPO) Generation of radical species was accompanied by the enzyme inactivation A superoxide scavenger, superoxide dismutase, did not protect the enzyme However, a thiyl radical scavenger, thioredoxin, prevented the inactivation It was concluded that the thiyl radical of the complex-bound dihydrolipoate induces the inactivation by 1e– oxidation of the 2-oxo acid dehydrogenase catalytic intermediate A product of this oxidation, the DMPO-trapped radical fragment of the 2-oxo acid substrate, inactivates the first component of the complex The inactivation prevents transformation of the 2-oxo acids in the absence of terminal substrate, NAD+ The self-regulation is modulated by thioredoxin which alleviates the adverse effect of the dihydrolipoate intermediate, thus stimulating production of reactive oxygen species by the complexes The data point to a dual pro-oxidant action of the complex-bound dihydrolipoate, propagated through the first and third component enzymes and controlled by thioredoxin and the (NAD+ + NADH) pool The 2-oxo acid dehydrogenase complexes are key mitochondrial enzymes functioning at branch points of metabolism They catalyze irreversible oxidation of 2-oxo- acids (pyruvate, 2-oxoglutarate or branched chain 2-oxoacids) yielding CO2, acyl-CoAs and NADH via reactions 1–5: Keywords: dihydrolipoate; 2-oxo acid dehydrogenase complex; reactive oxygen species; thiyl radical; thioredoxin Correspondence to V Bunik, A.N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia Tel.: + 095 939 14-56, Fax: + 095 939 31 81, E-mail: bunik@genebee.msu.su Abbreviations: E1, 2-oxo acid dehydrogenase; E2, dihydrolipoamide acyltransferase; E3, dihydrolipoamide dehydrogenase; DTPA, diethylenetriaminepentaacetic acid; DMPO, 5,5¢-dimethyl-1-pyrroline-N-oxide; MNP, 2-methyl-2-nitrosopropane; PBN, a-phenyl-N-tert-butylnitrone; POBN, a-(4-pyridyl-1-oxide)-N-tert-butylnitrone; ROS, reactive oxygen species; SOD, superoxide dismutase; ThDP, thiamin diphosphate (Received 31 May 2002, accepted 23 August 2002) Ó FEBS 2002 Radicals upon oxidation of 2-oxo acids (Eur J Biochem 269) 5005 Depending on the particular 2-oxo acid, R- is CH3-(pyruvate), HOOC-(CH2)2- (2-oxoglutarate), CH3-CH(CH)3CH2- (2-oxoisovaleriate) Multiple copies of the substratespecific 2-oxo acid dehydrogenase (E1), dihydrolipoamide acyltranferase (E2), and dihydrolipoamide dehydrogenase (E3) are organized in highly ordered structures [1–4] E1 catalyzes the rate-limiting step of the whole process [5–7] and the active sites are coupled through the interacting network of the lipoyl moieties [3,8] The two electrons in the catalytic intermediate of reactions and 5, the 2e) reduced E3, initially were thought to be distributed between flavin semiquinone and sulfur radical [9,10] However, at low temperatures this enzyme form is EPR-silent [11], and an internal charge transfer complex between a thiolate anion and oxidized FAD [12,13] is currently accepted as the best description Although the physiological process of the 2-oxo acid oxidative decarboxylation (Reactions 1–5) occurs through 2e– transfers, a number of related reactions involve radical species as intermediates The 1e– acceptor, ferricyanide, is able to oxidize catalytic intermediates formed by all the components of the 2-oxo acid dehydrogenase complexes In pyruvate:ferredoxin oxidoreductases [14–16] and in chemical models [17,18], hydroxyethylthiamine diphosphate (a product of reaction with pyruvate) and its analogs were shown to undergo oxidation via a thiazolium radical Redox reactions involving radical species were demonstrated for free and E3-bound FAD, including model reaction with the lipoic acid radical [19,20] and the reduction of oxygen to superoxide anion radical [21–23] Involvement of such processes in catalysis by the 2-oxo acid dehydrogenase complexes has not been previously investigated However, several considerations point to their regulatory potential First of all, the high catalytic power and the significant contribution of E3 to the total flavin content of mitochondria [24] suggest that the potential input of complex-bound E3 to the mitochondrial production of reactive oxygen species (ROS) should not be neglected ROS have attracted increasing attention as cellular messengers involved in differentiation, apoptosis and aging [25–27] Further, treatment of cells with H2O2 results in selective oxidation of the 2-oxo acid dehydrogenase complexes [28], that could also take place in vivo upon site-specific production of ROS via the complex-bound FAD In vitro and in situ inactivation of the 2-oxo acid dehydrogenase complexes upon addition of organic hydroperoxides [29–31] or a superoxide anion radical-generating system [32,33] correlate with specific targeting and/or impaired function of the complexes observed in many disorders linked to mitochondrial and cell damage These disorders include poisoning with environmental toxicants [34,35], Alzheimer’s [36,37] and Parkinson’s [38] diseases, Wernicke–Korsakoff syndrome [39] and others However, the mechanisms of oxidative damage of the complexes and cellular protection against this damage are not properly understood Because the redox state of cellular thiols and disulfides is an important factor in cellular protection against oxidative damage, we suggested that the 2-oxo acid dehydrogenase complexes may be significant not only as catalytic systems, but also as microcompartments of important biological thiols, lipoate and CoA The oligomeric complex cores form an inner cavity for CoA Depending on the source and type, the cores consist of 24 (cube) or 60 (pentagonal dodeca- hedron) E2 subunits, with each E2 subunit bearing up to three lipoate residues [2,3] In vitro, more than a half of the lipoyl residues of the E2 oligomer may be removed without significant change in the overall activity [40–42] If the residues function only as catalytic intermediates, the reason for their abundance is not clear However, in view of the antioxidant function of lipoate [43–45], its compartmentalization within the complexes may be important for cellular redox homeostasis through thiol-disulfide interchange Indeed, we have identified a flow of redox equivalents between the complexes and the medium by means of thioldisulfide exchange reactions involving the dihydrolipoate intermediate and cellular thiol-disulfide oxidoreductase, thioredoxin [46–48] Further study showed that the first component of the complexes is inactivated under increased steady-state concentration of the dihydrolipoate intermediate [49] This was surprising, as thiols are usually protective rather than inactivating However, the pro-oxidant action of dihydrolipoate is known [43–45,50,51] and could be involved in the inactivation The present study was undertaken to investigate the relationship between the free radical chemistry and function of the 2-oxo acid dehydrogenase complexes EPR and spin traps were used to study the complex-catalyzed reactions under a variety of conditions, including depletion of oxygen, presence of specific radical scavengers, and selective inactivation of the complex components We report that the 2-oxo acid dehydrogenase complexes produce several radical species of regulatory significance MATERIALS AND METHODS CoA, diphenyliodonium chloride, succinyl-CoA, b-D-glucose, N-ethyl maleimide, R,S-lipoamide, MNP, DTPA, glutathione disulfide, copper-zinc superoxide dismutase (from bovine erythrocytes, 3500 mg)1), glucose oxidase (from Aspergillus niger, 250 mg)1), cytochrome c (from horse heart, type VI) were from Sigma (Deisenhofen, Germany) 2-Oxoglutarate and pyruvate were from Merck (Darmstadt, Germany) DMPO, PBN, POBN and lucigenin were from Molecular Probes (Leiden, the Netherlands) Catalase (from bovine liver, 260 000 mg)1), ThDP, NAD+, and NADH were from Roche Molecular Biochemicals (Mannheim, Germany) Recombinant thioredoxin from Escherichia coli was from Calbiochem-Novabiochem GmbH (Bad Soden, Germany) R,S-Dihydrolipoamide was obtained from R,S-lipoamide by sodium borohydride reduction as in [47] Enzyme isolation, assays and modification 2-oxoglutarate and pyruvate dehydrogenase complexes from pig heart were isolated according to [52] with the modifications described earlier [46] The pyruvate dehydrogenase complex from E coli was isolated as in [53] Overall, E1 and E3 enzymatic activities were assayed spectrophotometrically [49] by NAD+ reduction, ferricyanide reduction and NADH oxidation, respectively The E2 or E3 components of the 2-oxoglutarate dehydrogenase complex were selectively modified at room temperature with freshly prepared solutions of N-ethyl maleimide or diphenyliodonium chloride according to [54] or [47] N-Ethyl maleimide (0.3 mM, final concentration) was added to the Ó FEBS 2002 5006 V I Bunik and C Sievers (Eur J Biochem 269) mixture of complex (9 mgỈmL)1) containing 2-oxoglutarate, ThDP, MgCl2 and NAD+ (1 mM each) After 20 min, the complex activity was not measurable, while activities of the E1 and E3 components were unchanged Diphenyliodonium chloride (4 mM, final concentration) was added to the mixture of complex (9 mgỈmL)1) with NADH (2 mM) in 0.1 M potassium phosphate buffer, pH 7.0 The residual activity of the E3 component after one hour of modification was no more than 4% of its initial value The modified complexes were separated from the low molecular mass compounds by desalting on a HiTrapTM mL column (Pharmacia, Uppsala, Sweden) in 0.1 M potassium phosphate, pH 7.0 EPR spectroscopy Room temperature EPR spectra were recorded in a quartz flat EPR sample cell at X-Band using a Bruker 300E EPR spectrometer (Karlsruhe, Germany) The spectrometer was operated at modulation amplitude mT, modulation frequency 100 kHz, microwave energy 20 mW The reactions took place in 0.05 M potassium phosphate buffer, pH 7.0 The stock solutions of MNP, DMPO, POBN and PBN were freshly prepared and kept protected from light Controls in each experiment indicated that none of the components alone produced the EPR signals studied Model reactions of thiyl radical trapping PBN took place in a reaction medium containing excess dithiothreitol and glutathione in the presence of Ce4+, as the latter is known to oxidize thiol groups through thiyl radicals FAD radicals were trapped by PBN in the reaction medium containing excess FAD in the presence of dithionite Anaerobic conditions were created either enzymatically or in a Weidner glove box (Hardegsen, Germany) connected to the MBraun GmbH H2O/O2-Analyzer and Inert gas-System (Garching, Germany), with the residual O2 pressure in the glove box not exceeding p.p.m To remove oxygen enzymatically, the reaction mixtures were preincubated for with the oxygen-scavenging system including glucose oxidase (10 mL)1), glucose (0.3 M) and catalase (26 mL)1) Distribution of the EPR signal between the protein and nonprotein fractions was investigated after precipitation of 2-oxo acid dehydrogenase complex by addition of 0.2 vol of 35% polyethylenglycol 6000 and solid ammonium sulfate to 80% saturation In anaerobic experiments, manipulations of the samples were performed in the glove box The precipitating agents were added to the probe after recording its EPR spectrum The precipitated protein was centrifuged for several minutes in a sealed Eppendorf tube, the pellet washed with saturated ammonium sulfate and the centrifugation repeated A stable and measurable EPR signal to compare the reactions in the presence and absence of O2 under otherwise equal conditions was achieved by employing high initial concentration of the 2-oxo acid dehydrogenase complexes (30–200 mgỈmL)1), so that the concentrations of substrates were comparable to that of the protein redox centers Lucigenin-dependent fluorescence This was measured with a Berthold LB 9505C Luminometer (Germany) Reaction mixtures of mL contained 2.5 mgỈmL)1 2-oxoglutarate dehydrogenase complex, 27 lM lucigenin, mM CoA, mM 2-oxoglutarate and 0.1 mM DTPA The readings for substrates and for the enzyme complex with lucigenin without substrates were used as blanks Ferricytochrome c reduction This was monitored at a cytochrome concentration of 16 lM by the increase at 550 nm due to formation of ferrocytochrome c, using a molar extinction coefficient of 20000 M)1Ỉcm)1 The reaction was carried out in 0.1 M potassium phosphate buffer, pH 7.0, in the presence of catalase (0.01 mgỈmL)1), EDTA (1 mM) and 2-oxoglutarate dehydrogenase complex (1–2 mgỈmL)1, specific activity in the NAD+ reduction 2–3 lmolỈmin)1Ỉmg)1) catalyzing transformation of either 2-oxoglutarate and CoA (2 mM each) or NADH (8 mM) The same mixture was used as a blank except that SOD (0.016 mgỈmL)1) was added Reactions were started with CoA or NADH Under these conditions initial rates of cytochrome c reduction in the presence of SOD were no more than 20% of those obtained without SOD Enzyme inactivation studies Time-dependent changes in the activity of the enzyme complex upon preincubation with its substrates and/or products was studied during a 20-min preincubation period in 0.1 M potassium phosphate buffer, pH 7.0, at the following final concentrations: enzyme complex, mgỈmL)1; 2-oxoglutarate, mM; CoA, 0.05 mM; succinyl-CoA, 0.3 mM; dihydrolipoate, 0.3 mM; NADH, 10 mM Samples were withdrawn at various times during the preincubation period and assayed for activity Ferricyanide-reductase activity of E1 was measured in all cases except those involving preincubation with NADH Because NADH interferes with the ferricyanide-dependent assay, overall NAD+-reductase activity was measured in the latter case and mM EDTA was added to stabilize the overall activity upon preincubation Under the conditions of the experiment, reversible inhibition of E3 by NADH did not affect the activity measured Thiol-containing compounds did not interfere with the ferricyanide assay due to the many-fold dilution of the preincubated mixture upon assay RESULTS Trapping of radical species in the course of reactions catalyzed by the 2-oxo acid dehydrogenase complexes The spin trapping technique allows one to detect unstable radical intermediates by converting them to more stable radicals Nitrone (PBN, POBN, DMPO) and nitroso (MNP) spin traps are known to react with short-lived radicals, resulting in relatively long-lived nitroxide radical adducts Together with the characteristic properties of the adducts formed, differential reactivity of spin traps to radicals enables selective trapping and identification of the original radical species The spin trap MNP is presumed to efficiently form adducts with catalytic radical intermediates, as it is small enough to reach enzyme active sites without major steric hindrance Aerobic incubation of MNP with the pyruvate or Ó FEBS 2002 Radicals upon oxidation of 2-oxo acids (Eur J Biochem 269) 5007 2-oxoglutarate dehydrogenase complexes and their respective 2-oxo acid substrates, CoA and NAD+, led to formation of MNP/•H, or t-butyl-hydronitroxide This product of oneelectron reduction [25] was detected from the four line EPR spectrum with aN ¼ aH ¼ 1.44 mT and its characteristic change observed in 50% D2O due to t-butyl-deuteronitroxide (aN ¼ 1.4 mT, aD ¼ 0.22 mT) Specific modification of the complex-bound FAD by diphenyliodonium chloride prevented the appearance of paramagnetic species Thus, the E3-bound FAD catalyzes the 1e– oxidation of the complex-bound dihydrolipoate intermediate by MNP MNP/•H was also formed in the reaction medium without NAD+ This shows that the reaction may either substitute or compete with the 2e– oxidation by the physiological substrate, NAD+ (Reactions 4–5) Aerobic incubation of the spin trap PBN with the 2-oxo acid dehydrogenase complexes and their substrates (2-oxoacid, CoA and NAD+) resulted in an EPR spectrum characteristic of a PBN adduct with radical species The EPR signal (spectrum 1, Fig 1) was that of a freely rotating nitroxide, with each line of the nitrogen triplet split into a doublet due to a hydrogen in the b-position to the nitrogen As in the reaction with MNP, the EPR signal persisted after omitting NAD+ from the reaction medium (Fig 1, spectrum 2) However, omitting either 2-oxo acid or CoA (i.e the components leading to the complex-bound dihydrolipoate) prevented the adduct formation Paramagnetic species were generated in an enzymedependent manner both in the presence of the 2-oxo acid, CoA (forward reaction) and when the 2-oxo acid dehydrogenase complexes catalyzed oxidation of NADH or external dihydrolipoamide (backward reaction) In any case, radicals were produced upon reduction of both the E2-bound dihydrolipoate and E3 The particular contribution of these components to the production of radicals was differentiated through their selective inactivation N-Ethyl maleimide modification of the lipoate residues of E2 led to complete loss of the overall activity (Reactions 1–5) due to E2 inactivation, while the E1 and E3 activities were fully preserved With this modified complex, no PBN adduct was observed in the presence of 2-oxo acid and CoA, but it did produce PBN adducts upon incubation with the E3 substrates, dihydrolipoate or NADH Modification of the tightly bound flavin cofactor of E3 with diphenyliodonium chloride inactivated E3 This complex gave no PBN adduct when incubated with either 2-oxo acid and CoA or NADH Thus, the E3-bound FAD was responsible for the formation of radical species at the expense of either complex-bound or free dihydrolipoate or NADH Action of the known radical scavengers and properties of the adducts obtained were studied to identify original radical species No EPR signal was detected in the presence of both SOD and catalase SOD alone blocked the appearance of the paramagnetic species during the first 20–25 of the reaction, but the EPR signal developed after the delay The delayed signal was inhibited by concomitant addition of SOD and the metal chelator DTPA These data show that initial PBN adducts are dependent on the superoxide anion radical generated in the system The delayed paramagnetic species are due to radicals arising in the presence of adventitious metal ions from H2O2, a product of the superoxide dismutation The conclusion is supported by the essential role of the E3-bound FAD in the radical production by the complexes, as FAD is known to reduce oxygen to superoxide [21–23] However, comparison of our experimental data to the data on the previously identified adducts (Table 1) shows that the stability and spectral characteristics of the PBN adducts detected in our system (N 1, 2) differ from those with reactive oxygen species (N 5, 6) The PBN (N 1) and POBN (N 17) adducts obtained in aerobic reactions with 2-oxo acids and CoA are similar to those known for thiyl radicals (N 13–15, 18,19) Precipitation of the protein after the aerobic reaction did not diminish the EPR signal which arose from the supernatant, indicating nonprotein PBN adducts Probably, the thiyl radical of CoA was trapped under these conditions An indirect relationship between the superoxide and PBN adducts is further supported by the fact that the hyperfine splitting constants of the aerobic PBN adducts formed in the forward (Table 1, N 1) and backward (Table 1, N 2) reactions differed Hence, secondary reactions with the initially produced superoxide or its PBN adduct must be invoked to explain formation of the stable PBN adducts characterized by the EPR spectra shown in Fig Direct detection of the superoxide anion radical produced by the complexes Fig EPR spectra of PBN adducts recorded under equal settings after 15 incubation of 2-oxoglutarate dehydrogenase complex (4 mg mL-1) with its substrates (2 mM each): (1) 2-oxoglutarate, CoA, NAD+ (2) 2-oxoglutarate, CoA Production of superoxide anion radical by the complexes was also examined by methods other than spin trapping Increased luminescence of lucigenin (bis-N-methylacridinium) upon its reaction with superoxide is used for specific detection of the latter in a number of biological systems [56] Up to a 10-fold increase in the luminescence occured upon incubation of the 2-oxoglutarate dehydrogenase complex with 2-oxoglutarate and CoA in the presence of lucigenin However, lucigenin itself may increase formation of superoxide in the presence of enzymes that are capable of catalyzing 1e– reduction of lucigenin directly [57], and E3 catalyzes 1e– reduction of various compounds [58–60] Therefore to quantify production of superoxide radical by E, 2-oxoglutarate or pyruvate, CoA, O2 E, NADH, O2 E, 2-oxoglutarate or pyruvate, CoA, anaerobic E, NADH, anaerobic E, pyruvate, CoA, O2 E, 2-oxoglutarate, CoA, anaerobic POBN DMPO Experimental system involving the 2-oxo acid dehydrogenase complexes PBN Spin trap Carbon-centered Alkyl (RC•) Matched to carboncentered radical Thiyl (RS•) Matched to thiyl radicals Thiyl (RS•) Thiyl (RS•) Alkyl (RC•) Alkoxy (RCO•) M urea 0.27–0.295 0.275–0.295 0.35 0.35 0.33 0.35 0.36–0.37 0.26–0.3 0.32 0.32 0.34–0.35 1.68 1.62 1.52–1.53 1.50–1.54 1.50 1.54 No adduct 1.58 1.59 of cysteine of glutathione of of of of of N-acetylcysteine CoA lipoate ethanol formate of cysteine of glutathione Methyl• 2.28–2.29 1.93 1.70–1.73 1.62–1.63 1.6 1.52 1.50–1.52 1.51 1.60 1.58 of protein Cys in • 0.36 0.24 0.26–0.27 0.23 0.28 2.24 1.53–1.56 1.48 1.51 1.51 1.53 1.56 1.58–1.65 1.62–1.63 1.56 1.56 1.56–1.57 0.33 ± 0.005 0.33 ± 0.01 0.33 ± 0.01 0.33 ± 0.01 aH (mT) OH OOH MethylO• MethylO• t-ButylO• of linoleic acid Methyl• of linoleic acid of DTT of glutathione of cysteine 1.59 ± 0.01 Matched to thiyl radicals Reactive oxygen species 1.6 ± 0.004 1.57 ± 0.01 Matched to alkyl radicals Matched to thiyl radicals • aN (mT) 1.56 ± 0.02 Previously identified radical adducts Matched to thiyl radicals Radical group not indicated transition metals with SH excess catalyze decay min Stable Transient species, stable at low substrate Transient species, stable at low substrate s to Unstable (min) Stable Stable Stable Stable Stable Stable min under oxidizing conditions; hours without O2 Stable Stable Stable Stable Stable Stable Stable Stability of adducts 16 17 18 19 20 21 10 11 12 13 14 15 N 22 23 24 25 26 27 28 87 50, 87, 88 84 85 86 67 77–79 78, 79 80 80 82 82 74, 80, 81 82, 83 This work This work 66, 84 Reference for identified radical adducts Table Comparison of the spin trap adducts obtained in the reactions catalyzed by the 2-oxoglutarate or pyruvate dehydrogenase complexes (E) with the known spin trap adducts in water medium Adducts with no decay during the time of experiment are referred to as stable 87 14 50, 68 14, 89, 90 91 5008 V I Bunik and C Sievers (Eur J Biochem 269) Ó FEBS 2002 Ó FEBS 2002 Radicals upon oxidation of 2-oxo acids (Eur J Biochem 269) 5009 the 2-oxoglutarate dehydrogenase complex, ferricytochrome c reduction [61,62] was used Both in the forward (with 2-oxoglutarate plus CoA, mM each) and backward (with NADH, mM) partial reactions, the specific activities of the complex in superoxide production measured as initial rates of SOD-inhibited ferricytochrome c reduction were about nmolỈmin)1Ỉmg)1 This corresponds to 0.3–0.4% of the overall NAD+-reductase activity of the complex (reactions 1–5) Radical species and the catalysis-induced inactivation of E1 Generation of ROS was documented in the current study under conditions shown to induce catalysis-dependent inactivation of the complexes [48,49] Therefore we examined the possibility that the inactivation (Fig 2A) was due to the superoxide anion radical generated by the system No protection from the aerobic inactivation was observed in the presence of SOD Besides, the complexes were inactivated by 2-oxo acid and CoA also under anaerobic conditions (Fig 2B) Thus, the enzyme inactivation was not caused by the ROS produced On the other hand, radical species were deteced in the absence of oxygen too (Fig 3) The spectrum obtained under anaerobic conditions created with glucose oxidase, glucose and catalase (Fig 3, spectrum 2) showed a weaker doublet at higher field, indicative of adduct decay during the field sweep As glucose is a known radical scavenger [63] and in our experiments it indeed decreased the signal of the PBN adducts already formed, a more detailed study was performed under anaerobic conditions created in a glove box Several properties of the anaerobic and aerobic PBN adducts differed First, unlike the aerobic spectra, the anaerobic ones exhibited no significant difference in hyperfine splitting constants for the forward (Table 1, N 3) and backward (Table 1, N 4) reactions This argued for the same radical species being trapped, in good agreement with the limited possibilities of secondary reactions in the absence of O2 Second, in contrast to the aerobic process, formation of Fig Inactivation of 2-oxoglutarate dehydrogenase complex in the presence of 2-oxoglutarate and CoA under aerobic (A) and anaerobic (B) conditions Concentration of substrates: 0.15 mM (1), 1.5 mM (2) Concentration of the complex used (9 mgỈmL)1  4.5 lM) corresponds to  0.3 mM sites for substrate and/or reducing equivalents (24E1 + 24E2 + 12E3-S2 + 12E3-FAD) Fig Spectra of PBN adducts obtained upon anaerobic incubation of 2-oxoglutarate dehydrogenase complex with 2-oxoglutarate and CoA 1: Anaerobiosis created in glove box, reaction took place for 18 in the presence of 2-oxoglutarate dehydrogenase complex (3 mgỈmL)1), 2-oxoglutarate and CoA (2 mM each), SOD (0.1 lM) and DTPA (0.1 mM); 2: after solutions were preincubated for several minutes with glucose oxidase, glucose and catalase, the reaction was started by mixing the 2-oxoglutarate dehydrogenase complex (9 mgỈmL)1) with the substrates (3 mM) and PBN and the spectrum was recorded immediately anaerobic adducts was not prevented by SOD and DTPA (Fig 3, spectrum 1) Third, the kinetics of the anaerobic and aerobic reactions were different Significantly higher intensity of the EPR signal was observed in the absence of O2 just after the start of the reaction However, these species rapidly disappeared when the substrates were in excess Under the same conditions, the aerobic species accumulated with time Both the initial accumulation and following disappearance of the anaerobic species were more pronounced at increased enzyme concentration If added substrates were limiting so that no continuous reduction of the complex redox centers occurred, the anaerobic PBN adducts were rather stable Fourth, the anaerobic reaction led to the PBN adduct being localized to the protein fraction, whereas the aerobic adduct under identical conditions was found in the supernatant (Fig 4) Different localizations of the EPR signal after the anaerobic and aerobic reactions with NADH indicated that a transient protein-bound radical intermediate, not detectable in the presence of oxygen, was trapped upon anaerobic reduction of the complex According to the backward catalytic process effected by NADH (reactions and 4), the protein-bound radical species (Fig 4B) could arise from either the E3 redox-active disulfide and FAD or E2-bound lipoate residues From those, only the latter may show no nitroxide immobilization, as the lipoyl-lysine side chains are mobile and protrude from the complex core [1–4] In particular, their essentially free rotation was observed upon spin labeling of the lipoyl 5010 V I Bunik and C Sievers (Eur J Biochem 269) Ó FEBS 2002 Fig DMPO spin trapping of anaerobic reaction medium containing 2-oxoglutarate dehydrogenase complex (9 mgỈmL-1), 2-oxoglutarate and CoA (4 mM each) Fig Localization of PBN adducts obtained upon incubation of E.coli pyruvate dehydrogenase complex (27 mgỈmL-1) with NADH (0.4 mM) under aerobic (A) and anaerobic (B) conditions 1: Before protein precipitation 2: Protein fraction 3: Non-protein fraction Fractionation is described in Materials and methods group with a maleimide carrying a nitroxide label [64,65] In contrast, the adducts with the E3 redox centers should show restricted rotation inherent in the nitroxides being localized to the protein interior Indeed, EPR spectra of the PBN adducts with the protein cysteine residues [66,67] as well as the spectrum obtained in our model reaction with PBN trapping intermediates of FAD reduction by dithionite, are qualitatively different from the spectra of the protein-bound adduct in Fig 4B Thus, catalysis-dependent kinetics of the anaerobic adduct, its protein localization, spectrum, and hyperfine splitting constants (Table 1, N and 4) characteristic of the PBN-trapped thiyl radicals (Table 1, N 13,14,15) allow us to conclude that under anaerobic conditions PBN traps radicals of the complex-bound dihydrolipoate residues Detection of the E2-bound dihydrolipoate thiyl radical correlated with the inactivation of the complexes by 2-oxo acid plus CoA (Fig 2B) In the absence of O2, both the inactivation (Fig 2B) and the EPR signal stability decreased with increasing concentration of substrates, i.e upon full reduction of the catalytic centers Dismutation of the thiyl radicals and reduction of their PBN adducts within the network of interacting lipoyl moieties of the E2 core provides a good explanation for these phenomena Considering possible mechanisms of the inactivation of the complex by the thiyl radical of dihydrolipoate, we took into account that (a) the overall activity (Fig 2) is decreased due to the irreversible inactivation of E1 [49], and (b) the thiyl radical of the lipoyl residue should efficiently interact with the E1 catalytic intermediate, as the lipoyl-bearing domain of E2 is designed for this interaction (reaction 2) In this case, 1e– oxidation of the carbanion in the E1 active site (a product of reaction 1) by highly electrophilic thiyl radical of the dihydrolipoyl residue of E2 had to be expected The reaction was confirmed by anaerobic spin trapping with DMPO This spin trap is known to be unreactive towards the lipoate radical species [50,68] Addition of DMPO to the anaerobic reaction mixture containing the 2-oxoglutarate dehydrogenase complex, 2-oxoglutarate and CoA resulted in the spectrum (Fig 5) characteristic of the carboncentered DMPO radical, known for the DMPO adducts with hydroxyalkyl (e.g ethanol) or formate radicals (Table 1, N 27 and 28) Because CO2 and the 1-hydroxy3-carboxypropyl moiety bound to ThDP are formed during the E1-catalyzed decarboxylation of 2-oxoglutarate (reaction 1), the spectrum and hyperfine splitting constants of the DMPO adduct obtained (Table 1, N 21) are consistent with the product of 1e– oxidation of the 2-oxoglutarate-ThDPE1 complex Under aerobic conditions, the thiyl radical of dihydrolipoate cannot be detected as its PBN adduct due to the concomitant presence of the superoxide anion radical, secondary reactions and poor spectral resolution of different PBN adducts However, several lines of evidence point to the presence of the thiyl radical in the aerobic system First, the complexes are inactivated by 2-oxo acid and CoA in the presence of O2 (Fig 2A) and the inactivation is not prevented by SOD In contrast, thioredoxin which is a known thiyl radical scavenger [69] fully protected the enzyme from the inactivation The thioredoxin protection was obvious not only when assaying the overall activity (reactions 1–5), but also upon generation of the paramagnetic species In the medium without thioredoxin, the EPR signal intensity reached saturation after the complex inactivation (10–15 of incubation with the substrates, Fig 2A) When thioredoxin was added, the initial rate of the EPR signal increase was the same, but no saturation was obvious during half-anhour Thus, as increased productivity of the complexes in Ó FEBS 2002 Radicals upon oxidation of 2-oxo acids (Eur J Biochem 269) 5011 Table Inactivation of 2-oxoglutarate dehydrogenase from pig heart upon incubation with its substrates and/or products (Reaction conditions are indicated in Materials and methods) Substrate(s) and/or product(s) added ki (min)1) 2-oxoglutarate Dihydrolipoamide CoA Succinyl-CoA NADH 2-oxoglutarate, CoA Succinyl-CoA, dihydrolipoamide NADH + 2-oxoglutarate 0.011 ± 0.005 0.009 ± 0.005 No inactivation No inactivation No inactivation 0.10 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 generation of radical species was observed in the presence of thioredoxin Another argument for the presence of an oxidizing sulfurcentered radical species under aerobic conditions is provided by the accompanying spectral changes of the complex Unchanged in the presence of 2-oxoglutarate, the spectrum exhibited a rapid decrease in absorbance at 450 and 350 nm after addition of CoA These changes are known to proceed upon reduction of E3 with dihydrolipoate However, a concomitant increase of comparable magnitude at 400 nm was also observed This change does not occur upon reduction of the isolated E3 or E3 bound to E2 lacking the lipoyl domain [70] On the other hand, it involves the spectral region characteristic of the three-electron bonds formed with sulfur participation [71] Similar spectral change at 400 nm, stable in time and insensitive to oxygen, was observed upon reaction of E3 with a strongly oxidizing • • radical Br2 – (but not with O2 –), which was attributed to formation of a disulfide radical anion followed by electron transfer to some other residue [20] Finally, a series of aerobic inactivation experiments support the proposed mechanism of the 2-oxo acid plus CoA-dependent inactivation of E1, involving the complexbound thiyl radical As seen from Table 2, the enzyme activity was not decreased in the presence of dihydrolipoate, NADH or succinyl-CoA This indicates that neither these products of the overall reaction, nor radical species formed in the incubation medium with dihydrolipoate or NADH inactivate E1 However, any combination of the substrates and/or products providing concomitant presence of the E1 catalytic intermediate and complex-bound dihydrolipoate (2-oxo acid + CoA; NADH + 2-oxoacid; dihydrolipoamide + succinyl-CoA) did lead to inactivation In particular, the appearance of the ThDP adduct with the substrate moiety during the complex-catalyzed succinyl-CoA hydrolysis [72] accounts for the inactivation by dihydrolipoamide in the presence of succinyl-CoA (Table 2) DISCUSSION Generation of radical species during catalysis by 2-oxo acid dehydrogenase multienzyme complexes has been documented in this work by EPR spectroscopy, ferricytochrome c reduction and lucigenin fluorescence The superoxide anion radical is produced upon the E3-catalyzed 1e– oxidation of the E2-bound dihydrolipoate intermediate The thiyl radical of the E2-bound dihydrolipoate formed in this reaction causes the 1e– oxidation of the 2-oxo acid adduct with ThDP through the site-directed action on E1 This results in the carbon-centered radical fragment in the E1 active site and the enzyme inactivation The inactivation is prevented by thioredoxin which is a known scavenger of thiyl radicals [69] The present work shows that production of radical species by the 2-oxo acid dehydrogenase complexes underlies several phenomena of regulatory significance: (a) sensitivity of the first component of the complex to the terminal step of the overall reaction (NADH or superoxide production), (b) thioredoxin-dependent modulation of this sensitivity, and (c) the 2-oxoacid, CoA-dependent generation of a cellular messenger, superoxide anion radical, which is increased in the presence of thioredoxin The isolated E3 component was reported to produce superoxide in the nonphysiological backward reaction of NADH oxidation [22] Under physiological conditions, this reaction is unlikely to contribute to the mitochondrial metabolism significantly: NADH is a strong inhibitor of E3 and should thus be much more efficiently oxidized by the enzymes of the respiratory chain, specifically designed for the NADH oxidation However, the current study shows that superoxide is catalytically produced by the complex-bound E3 in the physiological direction of 2-oxo acid oxidation and may take place concomitantly with the overall reaction (Fig 1, spectrum 1) In the presence of 2-oxo acid and CoA the complexes generate superoxide anion radical at a rate (1 nmolỈmin)1Ỉmg)1) comparable to that of the known superoxide producers: respiratory chain (0.3–6 nmolỈmin)1Ỉmg)1) [73], microsomes (0.7–4 nmolỈmin)1Ỉmg)1), or purified FAD-containing monooxygenase (3–6 nmolỈmin)1Ỉmg)1) [62] Inability of the FAD-modified complex to generate paramagnetic species rules out the direct oxidation of the accumulated complex-bound dihydrolipoate intermediate as a source of the superoxide and indicates that the integral complex structure is required for the radical species production The results of the current study and the site-specific • reactivity of O2 – also bear on consideration of the concept of metabolons, i.e intracellular compartmentalized functional units The regulatory potential of superoxide radical production by the 2-oxoglutarate dehydrogenase complex should greatly increase in the microenvironment of the citric acid cycle metabolon This implies close arrangement of the 2-oxoglutarate dehydrogenase complex and transition metal-dependent enzymes, such as aconitase and fumarase, • both rapidly reacting with O2 – Aconitase has been shown • to produce OH radicals upon interaction with the superoxide anion radical, which releases its iron(II) [74] Selective and simultaneous targeting of aconitase and 2-oxo acid dehydrogenase complexes under oxidative stress in mitochondria [34] favors the interpretation that the former interacts with the superoxide generated by the latter Apart from the induction of the removal of the transition metal from aconitase via superoxide production, the direct mobilization of transition metals by dihydrolipoate may add to its pro-oxidant action in vivo, as the ability of dihydrolipoate to mobilize ferritin-bound iron, possibly through a radical species, is known [44,51,75] Anaerobic reduction of the complexes in the presence of PBN revealed transient formation of dihydrolipoate thiyl radicals upon equilibration of the E2 and E3 redox centers Because of superoxide production, the aerobic system is too complicated to allow detection of thiyl radical adduction by Ó FEBS 2002 5012 V I Bunik and C Sievers (Eur J Biochem 269) PBN However, the 1e– oxidation of the reduced complex by oxygen implicates the redox equilibrium involving the E3- and E2-bound thiyl radicals and the E3-bound flavin semiquinone Under these circumstances, appearance of a strongly oxidizing thiyl radical of dihydrolipoate (E° of a number of RS•/RS– or RS•/RSH couples are approximately +0.75 or +1.33 V, respectively [71]) is supported by the enzyme spectral changes, the SOD- and O2-insensitive inactivation of the E1 catalytic intermediate (Fig 2, Table 2) and the protection by thioredoxin from such an inactivation Pro-oxidant action of thiyl radicals is avoided in the presence of thioredoxin, because the free radical species of thioredoxin, both disulfide and thiyl, are unreactive [69] In our system, migration of one electron between the dihydrolipoate thiyl radical and thioredoxin should preclude the radical-mediated modification of E1 and facilitate the dihydrolipoate radical dismutation A thioredoxin mutation which renders the protein sulfur radical more oxidizing (D30A, numbering of Chlamidomonas reinhardtii thioredoxin h) [69], leads to a decrease of the 2-oxoglutarate dehydrogenase complex activity, rather than the protection exhibited by the wild type thioredoxin [48] Such a modulation of the 1e– redox properties of thioredoxin by amino-acid substitution may explain the adverse action of some thioredoxins in the 2-oxo acid dehydrogenase system, as well as the specific and highly efficient protection by mitochondrial thioredoxin [48] As pointed out by Asmus [71], interaction of oxygen with thiyl radicals is considerably less efficient than previously thought This agrees with our observations that O2 does not prevent the E1 damage (Fig 2) and that the rates of decay of E1 activity and of the overall reaction are equal [49] Irreversible modification of the lipoate residues by RSOO• formation with the deleterious action of the latter on E1 should have caused a faster inactivation of the overall reaction compared to the partial E1 decay, because in this case both E1 and E2 were inactivated Thus, our data indicate that (a) oxygen addition to the complexbound dihydrolipoate radicals is less efficient than their interaction with E1, and (b) the dihydrolipoate radical intermediate survives long enough to be of regulatory significance By enabling E1 inactivation in response to the absence of E3 substrate, the dihydrolipoate radical intermediate transfers information from E3 to E1 As a result, superoxide production by the complexes is restricted unless thioredoxin is added Thioredoxin modulates the selfregulation of the complexes through abolition of the deleterious action of the dihydrolipoate thiyl radicals on E1 This provides an increased performance of the complexes not only in the overall reaction, but also in superoxide production Thus, the energy-providing oxidative decarboxylation of 2-oxoacids may influence mitochondrial metabolism also by means of the pro-oxidant action of the dihydrolipoate intermediate propagated through E3 (superoxide and thiyl radicals production) and E1 (catalytic intermediate radical production and inactivation) While formation of the intrinsic thiyl radical is deleterious for the 2-oxo acid oxidation, it also is an antioxidant defense mechanism, preventing the superoxide production by the complexes External regulation of these processes by a cellular thioldisulfide oxidoreductase, thioredoxin, points to the link between the complexes and thioredoxin-dependent pathways in mitochondria For instance, they may form an antioxidant defense system, analogous to recently discovered in mycobacteria where the 2-oxoglutarate dehydrogenase complex provides reducing equivalents to the peroxiredoxin alkyl hydroperoxide reductase through a thioredoxin-like protein [76] Multiple levels of regulation and sensitivity to integral parameters such as substrate concentrations and NADH/NAD+ ratio support biological significance of the complex-catalyzed radical reactions characterized in the present work ACKNOWLEDGEMENTS This work was partially supported by the Alexander von Humboldt Foundation The authors thank Prof U Weser and Dr H Hartman for their advices concerning the EPR measurements at the beginning of this work Critical reading of the manuscript by Prof J Mieyal and Prof A J L Cooper and the interest of Prof G Gibson to 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scavenger of thiyl radicals [69] The present work shows that production of radical species by the 2-oxo acid dehydrogenase complexes. .. reaction with the lipoic acid radical [19,20] and the reduction of oxygen to superoxide anion radical [21–23] Involvement of such processes in catalysis by the 2-oxo acid dehydrogenase complexes