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J Biol Chem, Vol. 275, Issue 19, 14064-14069, May 12, 2000
Mitochondrial Aconitase Is a Source of Hydroxyl Radical
AN ELECTRON SPIN RESONANCE INVESTIGATION*
Jeannette
Vásquez-Vivar §¶,
B.
Kalyanaraman§, and
Mary Claire
Kennedy
From the Department of Pathology, Cardiovascular
Research Center, the § Biophysics Research Institute, and
the Department of Biochemistry, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
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ABSTRACT |
Mitochondrial aconitase (m-aconitase) contains a
[4Fe-4S]2+ cluster in its active site that
catalyzes the stereospecific dehydration-rehydration of citrate to
isocitrate in the Krebs cycle. It has been proposed that the
[4Fe-4S]2+ aconitase is oxidized by superoxide,
generating the inactive [3Fe-4S]1+ aconitase. In this
reaction, the likely products are iron(II) and hydrogen peroxide.
Consequently, the inactivation of m-aconitase by superoxide may
increase the formation of hydroxyl radical (·OH) through the
Fenton reaction in mitochondria. In this work, evidence for the
generation of ·OH from the reaction of m-aconitase with
superoxide is provided using ESR spin trapping experiments with
5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and
-phenyl-N-tert-butylnitrone. Formation of
free ·OH was verified with the ·OH scavenger
Me2SO, which forms methyl radical upon reacting with ·OH. The addition of Me2SO to incubation mixtures
containing m-aconitase and xanthine/xanthine oxidase yielded methyl
radical, which was detected by ESR spin trapping. Methyl radical
formation was further confirmed using
[13C]Me2SO. Parallel low temperature ESR
experiments demonstrated that the generation of the
[3Fe-4S]1+ cluster increased with increasing additions of
superoxide to m-aconitase. This reaction was reversible, as >90% of
the initial aconitase activity was recovered upon treatment with
glutathione and iron(II). This mechanism presents a scenario in which
·OH may be continuously generated in the mitochondria.
 |
INTRODUCTION |
There is much debate in the literature on the relative importance
of hydroxyl radical (·OH) and peroxynitrite in free radical
pathology (1). Clarification of the mechanism centered on this subject
is of considerable importance, especially in mitochondria, cellular
organelles that are constantly exposed to low levels of superoxide
anion (2, 3). Several neurodegenerative diseases (e.g.
Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease
or amyotrophic lateral sclerosis) and aging have been linked to
mitochondrial oxidative damage that results in decreased mitochondrial
function (3, 4). However, in biological systems, it is nearly
impossible to associate a specific damage to a single oxidant. For
example, superoxide and nitric oxide (·NO) co-generated at very
low levels ( 10 8 M) in cells will form
peroxynitrite (ONOO ) via a nearly diffusion-controlled
reaction (5, 6). The toxicological significance of these species is
clearly dependent on cell type, the biological targets, and their
relationship to one another. One of the sensitive biological targets in
oxidative damage to mitochondria is aconitase, an iron-sulfur protein
that catalyzes the stereospecific dehydration-hydration of citrate to
isocitrate in the Krebs cycle (7).
Aconitase activity in mitochondria has been reported to be a sensitive
redox sensor of reactive oxygen and nitrogen species in cells (8-11).
Aconitase contains a cubane-type [4Fe-4S]2+ cluster in
its active site with three iron atoms bound to cysteinyl groups and
inorganic sulfur atoms and a fourth labile iron atom (Fe- ). This
Fe- is unique in that it is not bound to a protein cysteine, but
rather to a hydroxyl group of substrate and water (7). The labile
Fe- is released upon oxidation of the [4Fe-4S]2+
cluster with the concomitant formation of inactive
[3Fe-4S]1+ enzyme. Aconitase is inactivated rapidly by
superoxide (k 107
M 1 s 1) (12) in the presence and
absence of substrate and relatively slowly by peroxynitrite
(k 105 M 1
s 1) and ·NO (13, 14). However, the reaction
between aconitase and peroxynitrite is strongly inhibited by the
addition of substrate that binds to the enzyme with high affinity
(14).
It was recently proposed that the reaction between mitochondrial
aconitase (m-aconitase)1 and
superoxide plays a major role in mitochondrial oxidative damage
(15-17). During this reaction, it has been proposed that iron is
released from m-aconitase as iron(II) with the concomitant generation
of hydrogen peroxide. This facilitates the formation of "free"
hydroxyl radical in mitochondria. In the presence of intracellular
reducing agents (e.g. glutathione, ascorbate, and NADPH),
iron(II) is reincorporated into the inactive form of m-aconitase to
regenerate the active form. According to this proposal, hydroxyl radical should be continuously generated in mitochondria as a result of
the reaction between superoxide and aconitase. However, the
experimental verification of this intriguing mechanism has so far been lacking.
The objective of this study is to provide evidence, using ESR, for the
formation of hydroxyl radical and inactive [3Fe-4S]1+
species from the reaction between superoxide and purified m-aconitase. Direct low temperature ESR was used to quantify
[3Fe-4S]1+
species.2 Hydroxyl radical
was detected by ESR spin trapping using a novel phosphorylated spin
trap, 5-diethoxyphosphoryl-5-methylpyrroline N-oxide
(DEPMPO), and a loop-gap resonator, which makes it possible to obtain
ESR spectra using exceedingly small amounts of enzyme (19). Our results
indicate that the reaction between m-aconitase and superoxide releases
iron(II) from the [4Fe-4S]2+ cluster, which can
subsequently catalyze the formation of free hydroxyl radical. The
biological implications of these reactions are discussed.
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EXPERIMENTAL PROCEDURES |
Materials and General Methods--
Bovine heart mitochondrial
aconitase was purified according to published procedures (20). Protein
purity ( 95%) was determined by dye staining of SDS-polyacrylamide
gels. The cluster content was measured from iron, sulfide, and protein
content analyses as described previously (21, 22). Active enzyme was
prepared immediately before use by anaerobically incubating the protein with iron and dithionite. To remove excess reagent, the preparation was
desalted anaerobically on Sephadex G-50 columns equilibrated with
buffer (50 mM phosphate, pH 7.4). The same protocol was
followed for reactivation studies, except that dithiothreitol or
glutathione replaced dithionite as the reductant. Enzyme activity was
measured following the formation of cis-aconitate from
isocitrate at 240 nm, and the concentration was calculated using an
extinction coefficient of 3.6 mM 1
cm 1. The assay was performed at 25 °C in 90 mM Tris-HCl buffer, pH 8.0, containing 20 mM
DL-isocitrate. Rates of superoxide generation by a xanthine
(1-0.5 mM)/xanthine oxidase (0.1 unit/mg of solid) system
(Roche Molecular Biochemicals catalog no. 1048180) were determined by
following the reduction of ferricytochrome c (50 µM) at 550 nm, and the concentration was calculated using
an extinction coefficient of 21 mM 1
cm 1.
Electron Spin Resonance Measurements--
Direct ESR spectra
were obtained at 10-15 K using calibrated quartz ESR tubes. The
spectra were analyzed on a Varian E109 Century Series spectrometer
operating at 9.17-GHz and 100-kHz field modulation. A frequency counter
(EIP Model 548) was used to determine frequency, and a gaussmeter
(MH-110R Radiopan, NMR magnetometer) was used to determine field
positions. Instrumental conditions for detecting the
[3Fe-4S]1+ cluster were as follows: microwave power, 0.1 milliwatts; modulation amplitude, 5 G; time constant, 0.128 s; and scan
rate, 0.83 G/s. Quantification of spin concentration was carried out
using a 1.0 mM copper perchlorate standard by comparing the
double integral of the spectra and standard under similar conditions.
ESR spin trapping experiments were recorded at room temperature on a
Varian E109 spectrometer operating at 8.9-GHz and 100-kHz field
modulation equipped with a loop-gap resonator (18). The use of a
loop-gap resonator enabled the utilization of very small quantities of purified enzyme. ESR spin trapping experiments were analyzed at room
temperature, and the instrumental settings were as follows: microwave
power, 2 milliwatts; modulation amplitude, 1G; time constant, 0.128 s;
and scan rate, 1.67 to 0.83 G/s.
 |
RESULTS |
Spin Trapping of Hydroxyl Radical Formed from the Reaction between
Mitochondrial Aconitase and Superoxide--
Fig.
1A shows the DEPMPO-superoxide
adduct (DEPMPO-OOH) detected in 20-µl incubation mixtures of DEPMPO
(0.1 M) with the xanthine/xanthine oxidase system (78 µM O 2 min 1). Incubations of DEPMPO
with superoxide generated at higher rates (220 µM
min 1) produced a composite ESR spectrum consisting of
DEPMPO-OOH (62% contribution) and a DEPMPO-hydroxyl adduct (DEPMPO-OH)
(38% contribution) (Fig. 1B). The addition of active
aconitase to the above incubation mixtures abolished the formation of
DEPMPO-OOH and increased the intensity of DEPMPO-OH (Fig.
1C). The formation of DEPMPO-OH was instantaneous, and the
resulting ESR spectrum was stable for several minutes. However,
DEPMPO-OH increased with increasing concentrations of superoxide.
Mitochondrial aconitase (100 µM, 40-45 units mg protein 1) added to incubation mixtures in which
superoxide was generated at a rate of 220 µM
min 1 (Fig. 1B) enhanced the yield of DEPMPO-OH
by ~88% (Fig. 1C; cf. Fig. 1B). No
superoxide adduct was detected under these conditions, indicating that
m-aconitase out-competes DEPMPO for reaction with superoxide. Parallel
low temperature direct ESR measurements of the sample (Fig.
1C, inset) revealed that the enzyme was converted to the inactive [3Fe-4S]1+ aconitase.

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Fig. 1.
ESR spectra of incubations of DEPMPO with
superoxide in the absence and presence of m-aconitase. DEPMPO (0.1 M) was incubated in 50 mM phosphate buffer, pH
7.5, and xanthine (0.5 mM)/xanthine oxidase (0.25 units/ml)
(A) and xanthine (0.5 mM)/xanthine oxidase (0.62 units/ml) (B). In A, the dashed line
is a computer simulation fitted by considering two isomers of
DEPMPO-OOH (in gauss): Isomer 1, aN = 13.1, aH = 12.1, and aP = 49.7;
and Isomer 2, aN = 13.1, aH = 10, and aP = 49.5. In B, the dashed line is a computer simulation
fitted by considering two spin adducts and obtained using hyperfine
splitting constants (in gauss): DEPMPO-OH (38% contribution),
aN = 14, aH = 13.4, and
aP = 46.9; and DEPMPO-OOH (62% contribution),
Isomer 1, aN = 13.2, aH = 11.5, and aP = 50; and Isomer 2, aN = 13.1, aH = 13.2, and
aP = 48. C is the same as
B in the presence of 100 µM m-aconitase. The
dashed line is a computer simulation obtained using
hyperfine splitting constants (in gauss): aN = 14, aH = 13.1, and aP = 46.9. Inset, direct low temperature ESR of the
[3Fe-4S]1+ species. The spectrum is from a diluted sample
(5 µM) of C.
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The addition of the iron chelator Ferene®
(3-(2-pyridyl)-5,6-bis(2-[5-furylsulfonic acid])-1,2,4-triazine) to
incubation mixtures used to obtain Fig. 2
(trace A) abolished the formation of DEPMPO-OH (trace
B). The addition of desferrioxamine also abolished the generation
of DEPMPO-OH (data not shown). These results suggest that the
catalytically active iron concentration was increased during the
reaction of m-aconitase with superoxide. To determine that iron is
indeed released from the [4Fe-4S]2+ cluster as has been
previously shown (12), the effect of substrate on DEPMPO-OH yields was
investigated. The addition of saturating concentrations of
D-isocitrate (1 mM) to incubation mixtures of m-aconitase with superoxide diminished the yield of DEPMPO-OH by
~58% (Fig. 2, trace C; cf. trace
A). Control experiments demonstrated that isocitrate had no effect
on the formation of DEPMPO-OH from incubation mixtures containing iron
and xanthine/xanthine oxidase (data not shown). It is known that
binding of substrate stabilizes the [4Fe-4S]2+ cluster of
aconitase and thereby protects the enzyme against inactivation (7, 23).
It is therefore likely that the inhibitory effect of isocitrate on the
formation of DEPMPO-OH is due to a decreased rate of iron release from
the [4Fe-4S]2+ cluster.

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Fig. 2.
Iron released from m-aconitase catalyzes the
formation of hydroxyl radical. Trace A, m-aconitase
(100 µM) incubated with DEPMPO (0.1 M) in
phosphate buffer and xanthine (0.5 mM)/xanthine oxidase
(0.62 units/ml); B, same as trace A but
containing Ferene (10 mM); trace C, same as
trace A but in the presence of 1 mM isocitrate;
trace D, bolus addition of hydrogen peroxide (1 mM) to incubations containing m-aconitase (100 µM) and DEPMPO (0.1 M) in phosphate buffer.
Spectra are representative of at least two independent
experiments.
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To investigate the contribution of hydrogen peroxide formed from
superoxide on iron release from m-aconitase, spin trapping experiments
were performed with a bolus addition of hydrogen peroxide. The reaction
between hydrogen peroxide and m-aconitase generated DEPMPO-OH (Fig. 2,
trace D), which represented ~31% of the yield obtained
during the reaction of m-aconitase with superoxide. This result is
consistent with the relatively slower second-order rate constant for
the reaction of hydrogen peroxide with aconitase ( 103
M 1 s 1) (12) as compared with
the rate constant for the reaction between aconitase and superoxide
( 107 M 1 s 1).
Taken together, these results indicate that the reaction between m-aconitase and superoxide is primarily responsible for the release of
iron from the [4Fe-4S]2+ cluster, which subsequently
catalyzes the generation of hydroxyl radical.
Effect of Dimethyl Sulfoxide on Radical Adduct
Formation--
Because 5,5-dimethyl-1-pyrroline N-oxide
(DMPO)- and DEPMPO-hydroxyl adducts can be formed through several
mechanisms, additional experiments were performed using a hydroxyl
radical scavenger, dimethyl sulfoxide (Me2SO). It is well
known that hydroxyl radical reacts with Me2SO to form
methyl radical (·CH3); and therefore, the detection
of the DMPO- or DEPMPO-methyl adduct is a diagnostic indicator of free
hydroxyl radical formation (Reaction 1).
As shown in Fig. 3B, the
addition of Me2SO to incubation mixtures of m-aconitase and
superoxide produced a DEPMPO-carbon-centered adduct whose ESR
parameters are consistent with trapping of methyl radical. The identity
of the trapped radical was confirmed using 13C-labeled
Me2SO. Substitution of [12C]Me2SO
for [13C]Me2SO generated the
13C-labeled DEPMPO-methyl adduct,
DEPMPO-13CH3 (Fig. 3C). In
additional experiments, the spin trap
-phenyl-N-tert-butylnitrone (PBN), which forms
persistent spin adducts with carbon-centered radicals, was substituted
for DEPMPO. As shown in Fig. 3D, no ESR signal was detected
from the reaction between m-aconitase and superoxide in the presence of
PBN. This is consistent with the instability of PBN-oxygen-centered
adducts at physiological pH. However, the addition of Me2SO
to the incubation mixtures generated a spectrum corresponding to the
PBN-methyl adduct (Fig. 3E), and a 13C-labeled
PBN-methyl adduct was detected with
[13C]Me2SO (Fig. 3F). These
results demonstrate that the reaction between m-aconitase and
superoxide generates free hydroxyl radical.

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Fig. 3.
Generation of methyl radical from the
reaction of m-aconitase with superoxide in the presence of
Me2SO. A, m-aconitase (100 µM) incubated with xanthine (0.5 mM)/xanthine
oxidase (0.8 units/ml) in 50 mM phosphate buffer, pH 7.5, and DEPMPO (0.1 M). B, same as A plus
Me2SO (10%, v/v). Dashed line, computer
simulation obtained using hyperfine splitting constants (in gauss):
aN = 15.1, aH = 22, and
aP = 47.5. C, same as B
but in the presence of [13C]Me2SO.
Dashed line, computer simulation obtained using hyperfine
splitting constants (in gauss): aN = 15.1, aH = 22, aP = 47.5, and
a13C = 6.3. D, same as A
except PBN (50 mM) was substituted for DEPMPO.
E, same as D but containing Me2SO
(10%, v/v). Dashed line, computer simulation obtained using
hyperfine splitting constants (in gauss): aN = 15.8 and aH = 3.7. F, same as
D but containing [13C]Me2SO (10%,
v/v). Dashed line, computer simulation obtained using
hyperfine splitting constants (in gauss): aN = 15.8, aH = 3.7, and a13C = 3.7.
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Direct ESR Analysis of [3Fe-4S]1+ Cluster Formation
during Reversible Inactivation of Mitochondrial Aconitase by
Superoxide--
The characterization and quantitative analysis of
[3Fe-4S]1+ species generated from the reaction of
m-aconitase with superoxide were based on the comparative analysis with
standards obtained from the stoichiometric oxidation of m-aconitase
with ferricyanide (24) (Table I, part A).
Aliquots from incubation mixtures of m-aconitase with superoxide were
taken at different time points and immediately frozen in liquid
nitrogen to quench the reaction. As shown in Table I (part B),
incubation of m-aconitase (100 µM) with superoxide
generated at the rate of 95 µM min 1
quantitatively oxidized the [4Fe-4S]2+ cluster to the
[3Fe-4S]1+ species and inactivated the enzyme. After 5 min of incubation, m-aconitase was fully converted to the
[3Fe-4S]1+ form, and only a residual enzyme activity was
detected (Table I, part B). This result suggests that superoxide
stoichiometrically oxidizes the [4Fe-4S]2+ cluster to
form [3Fe-4S]1+. Note that the amount of
[3Fe-4S]1+ obtained was close to the maximal amount
expected from the fully oxidized enzyme. This indicates that oxidation
of the [4Fe-4S]2+ cluster by superoxide generates
iron(II) and [3Fe-4S]1+ and that a continued exposure of
the enzyme to superoxide and hydrogen peroxide does not disassemble the
cluster. As [3Fe-4S]1+ upon reduction can incorporate
iron(II) to regenerate the active [4Fe-4S]2+ form, the
reversibility of the oxidation of m-aconitase by superoxide was
investigated by examining the recovery of enzyme activity. The samples
used in low temperature experiments were thawed and reactivated by
anaerobic reduction with either glutathione (5 mM) or
dithiothreitol (10 mM) in the presence of iron(II). As shown in Table I (part B), reactivation of samples from a 5-min incubation of m-aconitase with superoxide led to the recovery of 90%
of the initial enzyme activity. This result demonstrates that
inactivation of m-aconitase generates [3Fe-4S]1+ and is
therefore a reversible reaction. The addition of isocitrate (1 mM) to incubation mixtures of m-aconitase did not prevent
superoxide-mediated oxidation of the cluster and loss of activity. This
result indicates that although isocitrate causes a decrease in the
formation of hydroxyl radical (Fig. 2), the extent of the inhibition is
not high enough to prevent the inactivation of the enzyme. Under these conditions, therefore, the recovery of enzyme activity was maximal (Table I, part B). As shown in Fig. 1, oxidation of m-aconitase by
superoxide generated hydroxyl radical, a strong oxidant that can
further oxidize amino acids critical for m-aconitase function. To
investigate whether or not hydroxyl radical generated from m-aconitase
contributes to the inactivation of m-aconitase by superoxide, the
effect of hydroxyl radical scavengers on inactivation and reactivation
of the enzyme was investigated. The addition of Me2SO
(10%, v/v) or DEPMPO (0.1 M) did not diminish
superoxide-mediated formation of inactive [3Fe-4S]1+
m-aconitase, and neither DEPMPO or Me2SO prevented enzyme
inactivation. A slight increase in m-aconitase activity was detected in
the presence of Me2SO, an effect that seems related to the
effect of Me2SO on enzyme stability. Taken together, these
results indicate that hydroxyl radical generated during the reaction of
m-aconitase with superoxide does not irreversibly inactivate the enzyme
(22, 25). Thus, in the presence of cellular reductants, it is likely that m-aconitase will recycle iron(II) and enhance the generation of
hydroxyl radical.
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Table I
Effect of superoxide and hydrogen peroxide on [3Fe-4S]1+
formation, activity, and reactivation of aconitase
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The effect of superoxide on [3Fe-4S]1+ formation and the
recovery of enzyme activity was further investigated in incubation mixtures containing m-aconitase at 10-fold lower concentrations than
described above (Table I, part C). Under this condition, m-aconitase
was oxidized to the [3Fe-4S]1+ species, but the recovery
of enzyme activity was lower than that previously detected (Table I,
part C; cf. part B). Hydrogen peroxide (1 mM)
also oxidized [4Fe-4S]2+ aconitase to form the
[3Fe-4S]1+ species. However, the recovery of enzyme
activity was much lower than that measured for superoxide under
similar incubation conditions (Table I, part D). These results indicate
that inactivation of m-aconitase by hydrogen peroxide generated from
superoxide dismutation or added as a bolus is due to oxidation of the
[4Fe-4S]2+ cluster, leading to protein destabilization.
 |
DISCUSSION |
Superoxide-mediated Redox Cycling of Mitochondrial
Aconitase--
The intriguing hypothesis on the reaction between
superoxide and [4Fe-4S]2+ clusters of dehydratases
leading to hydroxyl radical formation has been previously proposed (12,
26). However, to date, the experimental verification of this hypothesis
has not been provided. In this study, we present, for the first time,
ESR spin trapping evidence for the generation of hydroxyl radical
during superoxide-induced oxidation of m-aconitase (Figs. 1 and 3).
This work was made possible because of recent breakthroughs in ESR
technology and spin trap synthesis. First, the use of a loop-gap
resonator in spin trapping has made it feasible to obtain ESR spectra
from exceedingly small sample volumes ( 10 µl) (27). As a result,
we were able to perform a large number of experiments using highly
purified m-aconitase. Clearly, this would not be possible with
conventional ESR using cavity resonators since they use large sample
volumes. Second, the newly synthesized DEPMPO spin trap reacts with
superoxide to form a persistent DEPMPO-OOH adduct (28). Unlike
DMPO-OOH, the DEPMPO-OOH adduct does not spontaneously decay to form
the DEPMPO-OH adduct (18, 27). In this study, in which a mixture of
superoxide and hydroxyl radical was generated, the use of DEPMPO has
made spin trapping interpretations less confounding.
In addition to hydroxyl radical trapping, we demonstrated that the
[4Fe-4S]2+ aconitase is quantitatively oxidized to the
[3Fe-4S]1+ cluster by superoxide (Fig. 1 and Table I).
From monitoring the ESR-active [3Fe-4S]1+ aconitase, we
showed that superoxide releases only one iron atom from the
[4Fe-4S]2+ cluster (Table I), suggesting that the
reaction between superoxide and m-aconitase is stoichiometric. As shown
in Table I, this was also confirmed by measuring the activity recovery.
It is known that in the presence of cellular reductants such as thiols,
m-aconitase recycles iron by incorporating iron(II) (Scheme
I and Table I) (7, 16). Therefore, under
in vivo conditions, it is likely that aconitase undergoes
redox cycling between the [4Fe-4S]2+ and
[3Fe-4S]1+ forms. Another aspect of this reaction is that
inactivation of m-aconitase by superoxide is a reversible process and
that hydroxyl radical generated during the reaction does not destroy
the iron-sulfur cluster at the active site of the enzyme. This result
lends further support for using m-aconitase activity as a measure of
intracellular superoxide steady-state levels (8, 16). Oxidation of
m-aconitase by hydrogen peroxide also generates hydroxyl radical,
albeit at lower yields compared with superoxide-mediated oxidation of
aconitase. The reactivation of the enzyme was partial, however,
indicating that hydrogen peroxide may irreversibly inactivate the
enzyme by destabilizing the protein structure. Recently, it was
reported that cytosolic aconitase inactivation by hydrogen peroxide is reversible, although no data were provided (24).

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Scheme 1.
Mechanism of superoxide-mediated
generation of hydroxyl radical from m-aconitase (modified
from Ref. 16).
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Pathological Consequences--
Emerging data strongly suggest that
an increase in superoxide in mitochondria causes severe dysfunction (3,
4). Superoxide-mediated mitochondrial dysfunction may be explained by
enhanced formation of hydroxyl radical and aconitase inactivation. It
was reported that the anthracycline antibiotic doxorubicin causes a
rapid inactivation of m-aconitase in cardiomyocytes (29). In this
system, the superoxide dismutase mimetic manganese(III)
tetrakis-(4-benzoic acid) porphyrin inhibited aconitase inactivation,
whereas the nitric-oxide synthase inhibitor
Nw-nitro-L-arginine methyl ester
exacerbated aconitase inactivation (28). These data reveal that
conditions that lower intracellular nitric oxide enhance
m-aconitase activation, suggesting that superoxide is more
potent than peroxynitrite in the inactivation of m-aconitase (28).
The source of free iron in oxidative cellular pathology has not been
determined. It is likely that the reaction of m-aconitase with
superoxide releases iron, which, in turn, may increase the concentration of low molecular mass iron complexes with citrate (29) or
ATP or ADP (30). These iron complexes and iron bound to DNA (31)
represent a redox-active pool of iron capable of catalyzing free
radical reactions including the site-specific generation of hydroxyl
radical. In this study, we have shown that m-aconitase is a plausible
physiological source of free iron during oxidative stress resulting
from increased superoxide formation.
Summary--
We have demonstrated that the reaction between
m-aconitase and superoxide generates free hydroxyl radical. Superoxide
releases iron(II) from the [4Fe-4S]2+ aconitase, forming
the inactive [3Fe-4S]1+ aconitase and hydrogen peroxide,
thus facilitating hydroxyl radical formation by the Fenton reaction. It
is likely that the reaction between superoxide and m-aconitase may
enhance mitochondrial oxidative damage associated with the
pathophysiology of several chronic neurodegenerative and cardiovascular diseases.
 |
ACKNOWLEDGEMENT |
We thank Dr. William E. Antholine for
assistance in low temperature ESR experiments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants RR01008, GM51831, CA77822, and HL47250 and by American Heart Association Grant-in-aid 9950629N.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprints should be addressed:
Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI
53226. Tel.: 414-456-8095; Fax: 414-456-6515; E-mail:
jvvivar@mcw.edu.
2
Note that [3Fe-4S]1+ is
paramagnetic and hence ESR-active, whereas [4Fe-4S]2+ is
diamagnetic and ESR-inactive.
 |
ABBREVIATIONS |
The abbreviations used are:
m-aconitase, mitochondrial aconitase;
DEPMPO, 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide;
DMPO, 5,5-dimethyl-1-pyrroline N-oxide;
PBN, -phenyl-N-tert-butylnitrone.
 |
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