In the presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide anion radical was detected.. It was concluded that the thiyl radical of the complex-bound dihydrolip
Trang 1Inactivation of the 2-oxo acid dehydrogenase complexes
upon generation of intrinsic radical species
Victoria I Bunik1and Christian Sievers2
1
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
fluor-escence 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
protein-bound radical concluded to be the thiyl radical of the
complex-bound dihydrolipoate was trapped by
a-phenyl-N-tert-butylnitrone Another, carbon-centered, radical was
trapped in anaerobic reaction of the complex with
2-oxo-glutarate and CoAby 5,5¢-dimethyl-1-pyrroline-N-oxide
(DMPO) Generation of radical species was accompanied by
the enzyme inactivation Asuperoxide 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 Apro-duct 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 alle-viates 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 thio-redoxin and the (NAD++ NADH) pool
Keywords: dihydrolipoate; 2-oxo acid dehydrogenase com-plex; reactive oxygen species; thiyl radical; thioredoxin
The 2-oxo acid dehydrogenase complexes are key
mito-chondrial enzymes functioning at branch points of
metabolism They catalyze irreversible oxidation of
acids (pyruvate, 2-oxoglutarate or branched chain 2-oxo-acids) yielding CO2, acyl-CoAs and NADH via reactions 1–5:
Correspondence to V Bunik, A.N Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia Tel.: + 7 095 939 14-56, Fax: + 7 095 939 31 81, E-mail: bunik@genebee.msu.su
Abbreviations: E1, 2-oxo acid dehydrogenase; E2, dihydrolipoamide acyltransferase; E3, dihydrolipoamide dehydrogenase; DTPA, diethylene-triaminepentaacetic 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)
Trang 2Depending on the particular 2-oxo acid, R- is CH3
-(pyru-vate), HOOC-(CH2)2- (2-oxoglutarate), CH3-CH(CH)3
-CH2- (2-oxoisovaleriate) Multiple copies of the
substrate-specific 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 4 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
chemi-cal models [17,18], hydroxyethylthiamine diphosphate (a
product of reaction 1 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 compartmentali-zation 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 thiol-disulfide 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 intermedi-ate [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 under-taken 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
M A T E R I A L S A N D M E T H O D S
CoA, diphenyliodonium chloride, succinyl-CoA, b-D-glu-cose, N-ethyl maleimide, R,S-lipoamide, MNP, DTPA, glutathione disulfide, copper-zinc superoxide dismutase (from bovine erythrocytes, 3500 UÆmg)1), glucose oxidase (from Aspergillus niger, 250 UÆmg)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 UÆmg)1), ThDP, NAD+, and NADH were from Roche Molecular Biochemi-cals (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 dehy-drogenase complex from E coli was isolated as in [53] Overall, E1 and E3 enzymatic activities were assayed spectrophotometrically [49] by NAD+reduction, ferricya-nide 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 diphe-nyliodonium chloride according to [54] or [47] N-Ethyl maleimide (0.3 mM, final concentration) was added to the
Trang 3mixture of complex (9 mgÆmL)1) containing 2-oxoglutarate,
ThDP, MgCl2and NAD+(1 mMeach) After 20 min, the
complex activity was not measurable, while activities of the
E1 and E3 components were unchanged
Diphenyliodo-nium chloride (4 mM, final concentration) was added to the
mixture of complex (9 mgÆmL)1) with NA DH (2 mM) in
0.1M 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 5 mL column
(Pharmacia, Uppsala, Sweden) in 0.1M potassium
phos-phate, 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 1 mT, modulation
frequency 100 kHz, microwave energy 20 mW The
reac-tions took place in 0.05M 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 2 p.p.m To remove oxygen enzymatically,
the reaction mixtures were preincubated for 5 min with the
oxygen-scavenging system including glucose oxidase
(10 UÆmL)1), glucose (0.3M) and catalase (26 UÆmL)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,
manipula-tions 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
centri-fugation repeated Astable 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 mgÆmL)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
Lumino-meter (Germany) Reaction mixtures of 1 mL contained
2.5 mgÆmL)1 2-oxoglutarate dehydrogenase complex,
27 lM lucigenin, 5 mM CoA, 5 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
Ferricytochromec 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 20000M )1Æcm)1 The reaction was carried out in 0.1M potassium phosphate buffer, pH 7.0, in the presence of catalase (0.01 mgÆmL)1), EDTA(1 mM) and 2-oxoglutarate dehydrogenase complex (1–2 mgÆmL)1, specific activity in the NAD+ reduction 2–3 lmolÆmin)1Æmg)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 mgÆmL)1) was added Reactions were started with CoAor 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 0.1M potassium phosphate buffer, pH 7.0, at the following final concentrations: enzyme complex,
3 mgÆmL)1; 2-oxoglutarate, 2 mM; CoA , 0.05 mM; succi-nyl-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 Ferricya-nide-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 1 mM EDTAwas 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
R E S U L T S
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
Trang 42-oxoglutarate dehydrogenase complexes and their
respect-ive 2-oxo acid substrates, CoAand NAD+, led to formation
of MNP/•H, or t-butyl-hydronitroxide This product of
one-electron reduction [25] was detected from the four line EPR
spectrum with aN¼ aH¼ 1.44 mT and its characteristic
change observed in 50% D2O due to
t-butyl-deuteronitrox-ide (aN¼ 1.4 mT, aD¼ 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-oxo-acid, CoAand 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
dihydro-lipoate) prevented the adduct formation
Paramagnetic species were generated in an
enzyme-dependent manner both in the presence of the 2-oxo acid,
CoA(forward reaction) and when the 2-oxo acid
dehy-drogenase 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
contribu-tion of these components to the produccontribu-tion 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 CoAor 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 min 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 CoAare 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 CoAwas 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, secon-dary 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 1
Direct detection of the superoxide anion radical produced by the complexes
Production of superoxide anion radical by the complexes was also examined by methods other than spin trapping Increased luminescence of lucigenin (bis-N-methylacridi-nium) 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 CoAin the presence of lucigenin However, lucigenin itself may increase formation of super-oxide 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
Fig 1 EPR spectra of PBN adducts recorded under equal settings after
15 min incubation of 2-oxoglutarate dehydrogenase complex
(4 mg mL -1 ) with its substrates (2 m M each): (1) 2-oxoglutarate, CoA,
NAD+(2) 2-oxoglutarate, CoA.
Trang 5Spin trap
aN
aH
O2
O2
• OH •OOH
1.53–1.56 1.48
77–79 78,
5 6
• )
1.51 1.51 1.53 1.56 0.35 0.35 0.33 0.35
80 80 82 82
7 8 9 10
• )
• )
1.56 1.56 1.56–1.57 1.6
0.32 0.32 0.34–
min min min
O2
13 14 15 16
O2
• )
1.50–1.52 1.51
84 85
18 19
• )
• )
1.52–1.53 1.50–1.54 1.50 1.54 No
1.68 1.62
min trans
min min
87 50,
87 14 50,
22 23 24 25 26
1.58 1.59
min not
27 28
Trang 6the 2-oxoglutarate dehydrogenase complex,
ferricyto-chrome c reduction [61,62] was used Both in the forward
(with 2-oxoglutarate plus CoA, 2 mMeach) and backward
(with NADH, 8 mM) partial reactions, the specific activities
of the complex in superoxide production measured as initial
rates of SOD-inhibited ferricytochrome c reduction were
about 1 nmolÆmin)1Æmg)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
exam-ined 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 CoAalso 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
hyper-fine 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
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 inten-sity of the EPR signal was observed in the absence of O2just 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 detect-able in the presence of oxygen, was trapped upon anaerobic reduction of the complex
According to the backward catalytic process effected by NADH (reactions 5 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 immobiliza-tion, 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
Fig 2 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 m M (1), 1.5 m M (2).
Concentration of the complex used (9 mgÆmL)1 4.5 l M )
corres-ponds to 0.3 m M sites for substrate and/or reducing equivalents
(24E1 + 24E2 + 12E3-S + 12E3-FA D).
Fig 3 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 min in the presence of 2-oxoglutarate dehydrogenase complex (3 mgÆmL)1), 2-oxoglutarate and CoA(2 m M each), SOD (0.1 l M ) and DTPA (0.1 m M ); 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 mgÆmL)1) with the substrates (3 m M ) and PBN and the spectrum was recorded immediately.
Trang 7group 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 3 and 4)
charac-teristic 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 CoAresulted
in the spectrum (Fig 5) characteristic of the carbon-centered DMPO radical, known for the DMPO adducts with hydroxyalkyl (e.g ethanol) or formate radicals (Table 1, N 27 and 28) Because CO2and the 1-hydroxy-3-carboxypropyl moiety bound to ThDP are formed during the E1-catalyzed decarboxylation of 2-oxoglutarate (reac-tion 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-ThDP-E1 complex
Under aerobic conditions, the thiyl radical of dihydro-lipoate 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 CoAin 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 assay-ing 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 min 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-an-hour Thus, as increased productivity of the complexes in
Fig 5 DMPO spin trapping of anaerobic reaction medium containing 2-oxoglutarate dehydrogenase complex (9 mgÆmL-1), 2-oxoglutarate and CoA (4 m M each).
Fig 4 Localization of PBN adducts obtained upon incubation of E.coli
pyruvate dehydrogenase complex(27 mgÆmL -1 ) with NADH (0.4 m M )
under aerobic (A) and anaerobic (B) conditions 1: Before protein
pre-cipitation 2: Protein fraction 3: Non-protein fraction Fractionation is
described in Materials and methods.
Trang 8generation of radical species was observed in the presence
of thioredoxin
Another argument for the presence of an oxidizing
sulfur-centered 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
complex-bound 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;
dihydrolipo-amide + succinyl-CoA) did lead to inactivation In
partic-ular, the appearance of the ThDP adduct with the substrate
moiety during the complex-catalyzed
succinyl-CoAhydro-lysis [72] accounts for the inactivation by dihydrolipoamide
in the presence of succinyl-CoA(Table 2)
D I S C U S S I O N
Generation of radical species during catalysis by 2-oxo acid
dehydrogenase multienzyme complexes has been
documen-ted 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 under-lies 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 genera-tion 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 cata-lytically produced by the complex-bound E3 in the physio-logical 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 CoAthe complexes gen-erate superoxide anion radical at a rate (1 nmolÆmin)1Æmg)1) comparable to that of the known superoxide producers: respiratory chain (0.3–6 nmolÆmin)1Æmg)1) [73], microsomes (0.7–4 nmolÆmin)1Æmg)1), or purified FAD-containing monooxygenase (3–6 nmolÆmin)1Æmg)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 func-tional 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 super-oxide anion radical, which releases its iron(II) [74] Selective and simultaneous targeting of aconitase and 2-oxo acid dehydrogenase complexes under oxidative stress in mito-chondria [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
Table 2 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 k i (min)1)
CoANo inactivation
Succinyl-CoANo inactivation
2-oxoglutarate, CoA0.10 ± 0.01
Succinyl-CoA, dihydrolipoamide 0.03 ± 0.01
NADH + 2-oxoglutarate 0.04 ± 0.01
Trang 9PBN 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
unreac-tive [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
Athiore-doxin 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
thiore-doxin by amino-acid substitution may explain the adverse
action of some thioredoxins in the 2-oxo acid
dehydroge-nase 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
O2does 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
complex-bound 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
inter-mediate transfers information from E3 to E1 As a result,
superoxide production by the complexes is restricted unless
thioredoxin is added Thioredoxin modulates the
self-regulation 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
thiol-disulfide 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 dehy-drogenase 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
A C K N O W L E D G E M E N T S
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 this investigation are greatly acknowledged.
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