Archives of Biochemistry and Biophysics 488 (2009) 6975

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Archives of Biochemistry and Biophysics

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Inhibition of succinate-linked respiration and complex II activity

by hydrogen peroxide
Michelle D. Moser, Satoshi Matsuzaki, Kenneth M. Humphries *
Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

a r t i c l e

i n f o

Article history:
Received 14 April 2009
and in revised form 11 June 2009
Available online 18 June 2009
Keywords:
Cardiac mitochondria
Complex II
Succinate dehydrogenase
Hydrogen peroxide
Oxaloacetate
Malate dehydrogenase
Free radicals

a b s t r a c t
Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxidant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological concentrations. Previous studies, however, have suggested that complex II (succinate dehydrogenase) is
sensitive to H2O2-mediated inhibition. Nevertheless, the effects of H2O2 on succinate-linked respiration
and complex II activity have not been examined in intact mitochondria. Results presented indicate that
H2O2 inhibits succinate-linked state 3 mitochondrial respiration in a concentration dependent manner.
H2O2 has no effect on complex II activity during state 2 respiration, but inhibits activity during state 3.
It was found that conditions which prevent oxaloacetate accumulation during state 3 respiration, such
as inclusion of rotenone, glutamate, or ATP, blunted the effect of H2O2 on succinate-linked respiration
and complex II activity. It is concluded that H2O2 inhibits succinate-linked respiration indirectly by sustaining and enhancing oxaloacetate-mediated inactivation of complex II.
2009 Elsevier Inc. All rights reserved.

Introduction
Complex II (succinate dehydrogenase, succinateubiquinone
oxidoreductase) is a multifunctional enzyme, catalyzing the oxidation of succinate to fumarate as part of the Krebs cycle and concurrently introducing electrons into the electron transport chain via
reduction of ubiquinone. As a source of electrons, complex II can
drive both oxidative phosphorylation and the complex I mediated
reduction of NAD to NADH via reverse electron ow [1,2]. Reverse
electron ow is also responsible, in part, for the production of reactive oxygen species produced in mitochondria respiring on succinate as an energy source [39]. Thus, regulation of complex II
activity is critical to both modulate its activity in response to energetic demands and also to regulate or limit ROS production.
The activity of complex II is regulated by numerous factors,
including both Krebs cycle and electron transport chain intermediates. The multifaceted regulation of complex II is most apparent in
isolated, respiring cardiac mitochondria. Complex II in isolated
mitochondria is found predominantly in an inactive state [10,11]
that is bound to oxaloacetate, a Krebs cycle intermediate and potent inhibitor of complex II activity [1113]. Complex II is activated
upon addition of succinate, malonate, or, to a lesser extent, NADH
linked substrates, to coupled mitochondria. These ligands activate
complex II by promoting dissociation of oxaloacetate. Disassocia* Corresponding author. Address: Free Radical Biology and Aging Research
Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma
City, OK 73104, USA. Fax: +1 405 271 1437.
E-mail address: Kenneth-Humphries@omrf.org (K.M. Humphries).
0003-9861/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2009.06.009

tion of oxaloacetate facilitates reduction of covalently bound FAD

by substrate or reduced ubiquinone [12,14]. Complex II is inactivated during state 3 (ADP-dependent) respiration, and reactivated
during state 4 (ADP-independent) respiration [10]. This cycling of
activity, based upon the metabolic status of the mitochondria, occurs as a function of oxaloacetate removal and production, ubiquinone reduction and oxidation, and the activation of complex II by
ATP [12,15]. In addition, complex II and complex I display inverse
regulatory properties whereby the heightened activity of one complex represses the other, a phenomenon likely due to competition
between the two complexes for available ubiquinone [12,14,16,
17].
Mitochondria produce pro-oxidants, such as superoxide and
H2O2, under normal conditions and increased amounts under conditions of oxidative stress. H2O2 is of special interest not only as a
deleterious byproduct of oxidative stress, but also as a regulator of
enzymatic activity. As a relatively mild oxidant, the majority of
mitochondrial enzyme activities are impervious to physiological
concentrations of H2O2 [1820]. However, enzymes that contain
redox sensitive prosthetic groups or reactive thiols in their active
sites display sensitivity to low concentrations of H2O2 either by direct oxidation or via the formation of proteinglutathione mixed
disuldes [21].
It has previously been shown that complex II is one of select few
mitochondrial enzymes that is inhibited by H2O2 upon treatment
of intact mitochondria [19] or synaptosomes [18], and that this
inhibition is reversible [19]. It has also been shown that complex
II activity is susceptible to inhibition by thiol-modifying agents,
such as N-ethylmaleimide [22,23]. Nevertheless, the mechanisms

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M.D. Moser et al. / Archives of Biochemistry and Biophysics 488 (2009) 6975

by which H2O2 inhibits complex II have not been examined. The

goals of the present study were to therefore to dene the mechanism(s) H2O2-mediated complex II inhibition, specically as a
function of the mitochondrial metabolic status, and determine
how H2O2 affects succinate-linked respiration.

Materials and methods

thenoyltriuoroacetone (TTFA) sensitive reduction of ubiquinone1 (50 lM) at 280 nm (e = 13,700 M 1 cm 1) [27]. The presence of
0.1% Triton X-100 completely blocked ubiquinone reduction by
complex I [28]. Malate dehydrogenase (MDH):mitochondria were
diluted to a nal concentration of 0.1 mg/mL in a buffer containing
25 mM MOPS pH 7.4, 0.1% Triton X-100, 5.0 mM malate and indicated concentrations of ATP. Malate dehydrogenase activity was
measured spectrophotometrically as the rate of NADH production
at 340 nm upon addition of 1.0 mM NAD+.

Isolation of cardiac mitochondria

NAD(P)H measurements
SpragueDawley rats were anesthetized with a mixture of ketamine, xylazine, and acepromazine (3:3:1) (0.50.75 mL/kg).
Hearts were excised immediately after full anesthetization and
placed into 20 mL of ice-cold buffer containing 210 mM mannitol,
70 mM sucrose, 1.0 mM EDTA, 5.0 mM MOPS, pH 7.4 (buffer A).
Hearts were perfused with 10 mL of buffer A and then minced with
scissors. This was followed by Polytron homogenization. The
homogenate was then spun at 500g for 5 min at 4 C, the supernatant collected and passed through cheese cloth, and spun again for
10 min at 5000g. The resulting mitochondrial pellet was resuspended in approximately 200 lL of buffer A and protein concentration determined by the BCA method (Pierce) using BSA as a
standard.
Mitochondrial respiration
Mitochondria were diluted to 0.25 mg/mL in 210 mM mannitol,
70 mM sucrose, 5.0 mM KH2PO4, and 10 mM MOPS, pH 7.4 (buffer
B) containing 10 mM succinate. Respiration was measured polarographically at 25 C with a Clark-style oxygen electrode (Instech) in
the presence or absence of 1.0 lM rotenone and indicated concentrations of H2O2. State 2 respiration is dened as the rate of oxygen
consumption in the presence of substrate prior to the addition of
ADP [24]. State 3 respiration was initiated by addition of ADP
(0.5 mM) at 2.0 min. Two hundred units of catalase (Sigma) was
added as indicated. The starting amount of molecular oxygen in
the 0.6 mL electrode chamber was based on the assumption that
265 nmol of molecular oxygen are dissolved per milliliter at atmospheric pressure and 20 C. A high concentration of succinate
(10 mM) was necessary to achieve maximal rates of state 3 respiration, particularly when oxygen consumption was measured in
the absence of rotenone.
H2O2 consumption
Mitochondria were diluted to 0.25 mg/mL in buffer B with
10 mM succinate, 1.0 lM rotenone, and H2O2 as indicated. State
3 respiration was initiated by addition of 0.5 mM ADP at 2.0 min.
At incremental times, mitochondria were diluted 1:20 into a buffer
containing 25 mM K2HPO4, pH 7.25 with 0.1% Triton X-100,
0.5 mM hydroxyphenylacetic acid and 2.0 U/mL horseradish peroxidase. H2O2 was measured uorometrically using 320 nm excitation/425 nm emission [25,26].
Enzyme activities
Mitochondria were diluted to 0.25 mg/mL in buffer B with
10 mM succinate, 1.0 lM rotenone, and H2O2 as indicated. State
3 respiration was initiated by addition of 0.5 mM ADP at 2.0 min.
At times indicated, samples were diluted 1:10 in a buffer containing 25 mM MOPS pH 7.8, 5.0 mM MgCl2, 2.0 lg/mL antimycin A,
and 0.1% Triton X-100. It was found that this detergent solubilization methodology effectively locked complex II into either the active or inactive state that was present in the intact mitochondria.
Complex II activity was measured spectrophotometrically by the

Mitochondria were diluted to 0.25 mg/mL in buffer B with

10 mM succinate, 1.0 lM rotenone, and H2O2 as indicated.
NAD(P)H autouorescence was measured using 340 nm excitation/460 nm emission [29]. NADH and NADPH autouorescence
are indistinguishable, and therefore measurements are referred
to as NAD(P)H.
HPLC analysis of NADH and NADPH
Mitochondria were diluted to 0.25 mg/mL in buffer B with
10 mM succinate and H2O2 as indicated. State 3 respiration was
initiated by addition of 0.5 mM ADP at 2.0 min, and rotenone
(1.0 lM) added at 4.0 min. At given points during the incubation,
an aliquot of sample was diluted 1:4 into 0.5 M KOH. Samples were
then centrifuged (10 min at 14,000g), and the supernatant analyzed by isocratic reverse-phase HPLC using with a mobile phase
of pH 5.0 10 mM KH2PO4 at a ow rate of 1.0 mL/min. NADH and
NADPH were monitored by uorescence detection (340 nm excitation/460 nm emission) and identication veried by comparison of
peak retention times to NADH and NADPH standards.
Statistical analysis
Data are presented as means SE. The data were evaluated utilizing a two-tailed t-test. Statistical signicance was assigned for
p 6 0.05, as indicated.
Results
The effects of H2O2 on succinate-linked respiration
To determine the effect of H2O2 on succinate-linked respiration,
cardiac mitochondria were isolated and respiration measured in
the presence or absence of H2O2. As shown in Fig. 1A, control cardiac mitochondria respiring on succinate demonstrate three
phases of state 3 (ADP-dependent) respiration. There is a fast initial
rate of oxygen consumption, followed by a slower steady state rate.
As most of the ADP is consumed, the rate of respiration once again
increases. The initial slowing of respiration is attributable to the
accumulation of the complex II inhibitor, oxaloacetate, during state
3 [1113], while the increase in the third phase is likely mediated
by reactivation of complex II by ATP [12,15]. H2O2 has no effect on
the fast, initial rate of state 3 respiration, but inhibits phase 2 state
3 respiration in a concentration dependent manner. H2O2 (50 lM)
inhibits the rate of state 3 respiration by 40%, but has no signicant
effect on state 2 or 4 respiration (Table 1). Cardiac mitochondria
have a low respiratory control ratio when utilizing succinate
(RCR = 2.1 0.1)1 as compared to mitochondria respiring on the
NADH linked substrates glutamate and malate (RCR P 7 for identical
samples). The low RCR was further exasperated by incubation with

1
Abbreviations used: RCR, respiratory control ratio; SDH, succinate dehydrogenase;
MDH, malate dehydrogenase; ROS, reactive oxygen species.

M.D. Moser et al. / Archives of Biochemistry and Biophysics 488 (2009) 6975

71

these experimental conditions. Under control conditions, rotenone

increased state 3 respiration by 69% and doubled the RCR as compared to respiration in its absence (Fig. 1B and Table 1). Interestingly, H2O2 had no effect on state 3, succinate-linked respiration,
in the presence of rotenone. The protection afforded by rotenone
was not due to increased rates of H2O2 clearance or antioxidant
capacity. The rate of consumption of 50 lM H2O2 by mitochondria,
in the presence or absence of rotenone, was found to be identical
and essentially complete by 15 min (11.2 nmol H2O2 min 1 mg 1
for mitochondria with succinate alone and 10.4 nmol
H2O2 min 1 mg 1 for mitochondria with succinate and rotenone).
Reversibility of H2O2-mediated inhibition and protection by ATP

Fig. 1. Hydrogen peroxide inhibits succinate-linked state 3 respiration but is

prevented by rotenone. Rat heart mitochondria (0.25 mg/mL) were incubated at
25 C in buffer B with 10 mM succinate and oxygen consumption was measured
polarographically with a Clark-style oxygen electrode. When H2O2 was present, it
was added at t = 0. (A) Mitochondria were incubated with 0 (black), 25 lM (blue), or
50 lM (red) H2O2 and state 3 respiration initiated upon addition of 0.5 mM ADP at
2.0 min. State 3 respiration demonstrates 3 distinct phases, as indicated. (B)
Mitochondria were incubated with 1.0 lM rotenone in the absence (solid) or
presence (hatched) of 50 lM H2O2 and state 3 respiration initiated by 0.5 mM ADP
at 2.0 min.

Table 1
Mitochondrial succinate-linked respiratory rates in the presence or absence of H2O2.
Rat heart mitochondria (0.25 mg/mL) were incubated at 25 C with 10 mM succinate
in the presence or absence of 50 lM H2O2 and/or 1.0 lM rotenone. Oxygen
consumption was measured polarographically with a Clark-style oxygen electrode
and state 3 respiration initiated by the addition of 0.5 mM ADP. Values are given as
nmol O2 min 1 mg 1 SE (n P 5 for each experimental condition).

Control
H2O2
Rotenone
Rotenone + H2O2

State 3

State 4

RCR

114.2 6.3
69.0 3.0
193.1 7.2
202.6 4.5

55.2 4.5
50.0 3.1
47.3 4.8
47.7 1.1

2.1 0.1
1.4 0.1
4.2 0.4
4.3 0.1

50 lM H2O2 (1.4 0.06), a function of decreased state 3 but not state

4 respiration.
Succinate-linked respiration was next examined in the presence
of 1.0 lM rotenone, a concentration of complex I inhibitor that was
found to completely block NADH-linked respiration. While rotenone can increase superoxide production during NADH-linked respiration [30], addition of the inhibitor to succinate-linked
respiration blocks superoxide production, and thus hydrogen peroxide production, by preventing reverse electron ow [8,31]. Thus
endogenous H2O2 production was not a confounding factor under

To demonstrate the reversibility of H2O2-mediated inhibition of

succinate-linked respiration, experiments were performed in
which catalase was added prior to ADP. As shown in Fig. 2, addition
of catalase to samples containing H2O2 results in an immediate upward deection in O2 concentration, indicating the rapid decomposition of H2O2 to H2O and O2. Importantly, this removal of H2O2
completely prevented decits in state 3 respiration.
The transient inhibition of H2O2 on succinate-linked respiration
was further characterized by experiments in which a second bolus
of ADP was added during state 4 respiration. Reinitiating state 3
respiration resulted in a rate of oxygen consumption that was
44% faster than the initial rate of state 3 (Fig. 3A) in control mitochondria. Importantly, reinitiating state 3 respiration resulted in
the same accelerated rate in both control and H2O2-treated mitochondria (Fig. 3A), thus demonstrating the effects of H2O2 on respiration are reversible. Furthermore, mitochondria in state 4 (ATP
present) were found to be resistant to further decreases in respiration upon addition of another bolus of 50 lM H2O2 prior to second
addition of ADP (data not shown). The reason for this, it was postulated, was that ATP present in state 4 acted to protect against
H2O2-mediated inhibition. This was conrmed by the experiment
in Fig. 3B, which demonstrates addition of ATP prior to state 3 increased the maximal rate of respiration and completely prevented
H2O2-mediated inhibition.
Complex II activity and H2O2
It has been previously reported that complex II is sensitive to
oxidative inactivation [19,32]. We therefore examined whether
H2O2-mediated inhibition of succinate-linked respiration was

Fig. 2. H2O2-induced inhibition of succinate-linked respiration is reversible by

catalase. Rat heart mitochondria were incubated in the presence (hatched and red)
or absence (solid) of 50 lM H2O2 added at t = 0. Catalase (200 U) was added (red
trace) at 1.75 min and state 3 respiration initiated by addition of 0.5 mM ADP at
2.0 min.

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M.D. Moser et al. / Archives of Biochemistry and Biophysics 488 (2009) 6975

Fig. 3. H2O2-induced inhibition of succinate-linked respiration is both reversed and

prevented by ATP. Mitochondria (0.25 mg/mL) were incubated in the presence
(hatched) or absence (solid) of 50 lM H2O2 and respiration measured as in Fig. 1.
(A) State 3 was initiated by addition of 0.5 mM ADP at 2.0 min, and then reinitiated
by addition of another bolus of 0.5 mM ADP at 11 min. (B) 0.5 mM ATP was added to
mitochondria, in the presence or absence of 50 lM H2O2, at t = 0 and state 3
initiated by addition of 0.5 mM ADP at 2.0 min.

accompanied by loss of complex II activity. It was found that in our

isolated cardiac mitochondria, in agreement with previous ndings, complex II is predominantly in the inactive state [10,11,33].
Complex II activity increased by 70% upon incubation of intact
mitochondria with succinate (state 2 respiration) and this activation was unaffected by 50 lM H2O2. Both control and H2O2-treated
samples showed a dramatic decrease in complex II activity upon
addition of ADP, a phenomenon attributable to the increase in oxaloacetate production during state 3 respiration [1113]. Noteworthy, the H2O2-treated sample demonstrated an enhanced
inactivation of complex II activity during state 3 respiration
(Fig. 4A), with activity 27% below control values after addition of
ADP (5.0 min time point). In control mitochondria, complex II
activity returns to the active state and temporally, this corresponded to the transition to state 4 respiration. Complex II activity
in H2O2-treated mitochondria also returns to the active state but at
an impaired rate that corresponded to the longer duration of state
3 conditions shown in Fig. 1.
Complex II activity was next examined in mitochondria respiring on succinate in the presence of rotenone (Fig. 4B). In control
mitochondria, rotenone largely prevented the inactivation of complex II during state 3 respiration. This phenomenon is attributable
to the maintained pool of reduced NADH during state 3 respiration,
the consequence of which is the impediment of the forward reaction catalyzed by malate dehydrogenase (MDH) to produce oxalo-

Fig. 4. Complex II activity is decreased during state 3 respiration and this is

exacerbated by H2O2, but prevented by rotenone. Mitochondria (0.25 mg/mL) were
incubated in the absence (dark bars) or presence (light bars) of 50 lM H2O2, added
at t = 0, as described in Fig. 1 and ADP (0.5 mM) added at 2.0 min to initiate state 3
respiration. At times indicated on the abscissa, 100 lL of sample was removed and
diluted 1:10 into a buffer containing 0.05% Triton X-100 and complex II activity
measured spectrophotometrically by monitoring ubuqinone-1 reduction. (A) Mitochondria were incubated in the presence of 10 mM succinate alone. Symbols ()
indicate statistical signicance (p < 0.05) between the indicated time points using a
student t-test. (B) Mitochondria were incubated in the presence of 10 mM succinate
and 1.0 lM rotenone. Values are means SE (n P 5 for each experimental condition). (C) Malate dehydrogenase was assayed, as described in Materials and
methods, with increasing concentrations of ATP as indicated. Traces represent
averages from experiments performed in triplicate.

acetate. Importantly, it was found that rotenone also blunted

inhibition of complex II activity by H2O2 (Fig. 4B). It should be
noted that rotenone did not completely prevent inactivation
of complex II during state 3 respiration in either control or

M.D. Moser et al. / Archives of Biochemistry and Biophysics 488 (2009) 6975

73

H2O2-treated mitochondria, indicating that a slight inactivation of

complex II is not sufcient to impede the maximal rate of succinate-linked respiration. The effect of ATP on complex II activity
was next examined. ATP, which prevented H2O2-induced loss of
state 3 respiration (Fig 3B), also largely prevented inactivation of
complex II in control mitochondria and H2O2-treated mitochondria
during state 3 respiration. Upon initiation of state 3 respiration in
the presence of ATP, complex II activity decreased only 17% in control mitochondria and 27% in H2O2-treated mitochondria as compared to basal activities in state 2. This is in contrast to a
decrease of over 60% in control mitochondria in the absence of
ATP. ATP prevents the accumulation of oxaloacetate catalyzed by
malate dehydrogenase [34], and activates complex II [12]. The effect ATP has on MDH activity is demonstrated in Fig. 4C in which
activity was assayed in the presence of increasing concentrations
of ATP. As the gure illustrates, the rate of oxaloacetate formation
followed rst order kinetics, with activity rapidly decreasing as
oxaloacetate accumulated. Importantly, addition of ATP inhibited
the rate of oxaloacetate production in a concentration dependent
manner.
Mechanism of H2O2-mediated complex II inactivation
Several lines of evidence suggest H2O2 is inhibiting succinatelinked respiration by a mechanism that induces increased inactivation of complex II activity during state 3 respiration, and not via direct oxidation of the enzyme. For example, complex II activity is
completely resistant to H2O2-mediated inhibition during state 2
respiration. In addition, both rotenone and ATP completely blocked
H2O2-mediated inhibition of state 3 respiration and largely prevented H2O2-mediated inactivation of complex II during state 3
respiration.
The effects that rotenone has on succinate-linked respiration
and complex II activity have been previously characterized. Rotenone prevents the accumulation of oxaloacetate during state 3 respiration by maintaining a large ratio of [NADH]/[NAD+] [3537],
thereby sustaining complex II in the active state. This suggests that
the effect of H2O2 on succinate-linked respiration is to enhance the
accumulation of oxaloacetate, thereby leading to depressed complex II activity. However, a direct assessment of oxaloacetate concentrations in isolated mitochondria is challenging. Because of the
unfavorable thermodynamics of the reaction catalyzed by MDH,
oxaloacetate production is extremely limited even in the presence
of excess malate [38]. Nevertheless, an indirect assessment of oxaloacetate involvement can be tested. Oxaloacetate, when glutamate is present, is a substrate for a transaminase that catalyzes
the conversion to a-ketoglutarate and aspartate [9]. Mitochondria
were therefore incubated with succinate and glutamate in the
presence or absence of 50 lM H2O2. A low concentration of glutamate (1.0 mM) was used, which on its own, was unable to support
state 3 respiration (Fig. 5A). However, this concentration of glutamate enhanced succinate-linked state 3 and prevented the second,
slow phase, of state 3 respiration that is normally present in control mitochondria (Fig. 5A). Importantly, glutamate completely
prevented H2O2-mediated inhibition of state 3 respiration
(Fig. 5A). Furthermore, it was found that glutamate prevents inactivation of complex II activity during state 3 respiration in control
mitochondria and attenuated the effect of H2O2 (Fig. 5B).
Modulation of NAD(P)H by H2O2
Our results suggest H2O2 is inhibiting succinate-linked respiration by modulating oxaloacetate-mediated inactivation of complex
II. Oxaloacetate production is catalyzed by malate dehydrogenase
(MDH), a reaction that occurs only under conditions of high ratios
of [NAD+]/[NADH] and [malate]/[oxaloacetate] [39]. The status of

Fig. 5. Glutamate prevents H2O2-mediated inhibition of succinate-linked respiration and decreases inactivation of complex II during state 3 respiration. (A)
Mitochondria (0.25 mg/mL) were incubated in the absence (black) or presence of
50 lM H2O2 (red) and respiration measured as in Fig. 1, with state 3 respiration
initiated at 2.0 min by addition of ADP (0.5 mM). Solid traces represent samples
respiring on 10 mM succinate alone, hatched lines had 10 mM succinate and
1.0 mM glutamate, and the blue trace had only 1.0 mM glutamate. (B) Mitochondria
(0.25 mg/mL) were incubated with 10 mM succinate and 1.0 mM glutamate in the
absence (dark bars) or presence (light bars) of 50 lM H2O2 added at t = 0. At times
indicated on the abscissa, an aliquot of sample was removed and complex II assayed
as in Fig. 4. Values are means SE (n P 3 for each experimental condition).

the NAD(P)H pool, as measured by autouorescence, can therefore

provide insight into conditions that promote oxaloacetate production. As shown in Fig. 6A, under control conditions, mitochondria
respiring on succinate show a marked decrease in NAD(P)H during
state 3 respiration, a condition that allows for oxaloacetate production. Treatment of mitochondria with H2O2 (50 lM) did not affect
the magnitude of NAD(P)H oxidation, but did extend the duration
of oxidative conditions. These extended oxidative conditions correspond to the lengthened duration of state 3 conditions.
Experiments were next performed in which rotenone was
added to mitochondria during state 3 respiration. Rotenone blocks
NADH consumption by complex I and prevents succinate-driven
reverse electron ow [2]. Thus, the increase in NAD(P)H upon addition of rotenone (Fig. 6B) during state 3 respiration is mediated by
the reduction of NAD+ by Krebs cycle dehydrogenases and prevention of consumption of NADH by complex I. In the presence of
H2O2, addition of rotenone during state 3 respiration increases
NAD(P)H in a rate similar to control mitochondria. However, upon
return to state 4 (ATP present) conditions, NAD(P)H decreased in a
time dependent manner. This rotenone-insensitive consumption of
NAD(P)H was contingent upon the presence of both ATP and H2O2.
Removal of H2O2 by catalase restored NAD(P)H levels to control

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M.D. Moser et al. / Archives of Biochemistry and Biophysics 488 (2009) 6975

Fig. 6. H2O2 induces rotenone-insensitive consumption of NAD(P)H in mitochondria respiring on succinate. Mitochondria (0.25 mg/mL) were incubated in the
absence (black traces) or presence of 50 lM H2O2 (red traces) as in Fig. 1. NAD(P)H
autouorescence was measured using 340 nm excitation/460 nm emission [29].
NADH and NADPH autouorescence are indistinguishable, and therefore measurements are referred to as NAD(P)H. (A) ADP (0.5 mM) was added at 2.0 min. (B) ADP
(0.5 mM) was added at 2.0 min, and then rotenone (1.0 lM) was added at 4.0 min
while mitochondria were still in state 3 respiration. Catalase (200 U) was added at
10 min.

conditions (Fig. 6B). This consumption of NAD(P)H is likely attributable to malate dehydrogenase catalyzing the consumption of
oxaloacetate to malate.
The uorescence measurements in Fig. 6 represent the sum of
autouorescence contributions from NADH and NADPH, although
the concentration of NADH is much higher than NADPH in mitochondria [29,40]. Nevertheless, it may be expected that H2O2-driven decrease in NAD(P)H uorescence is attributable to the
activities of glutathione reductase or thioredoxin reductase which
consume NADPH as part of the mitochondrial glutathione peroxidase and peroxiredoxin 3 H2O2 detoxication systems, respectively. NADH and NADPH were therefore extracted and analyzed
by reverse-phase HPLC to determine the redox status of each
nucleotide pool. Results indicate that the H2O2-driven decrease in
NAD(P)H autouorescence seen in Fig. 6B are due to a decrease
in NADH, while NADPH levels are unchanged (results now shown).
This result supports an MDH-dependent decrease in NADH, but
cannot conclusively exclude a contribution from a transhydrogenase reaction that replenishes NADPH at the expense of NADH.
Discussion
The present report demonstrates that H2O2 inhibits succinatelinked respiration. The data supports a mechanism whereby oxalo-

acetate production during state 3 leads to a sustained inactivation

of complex II. It has been previously shown that in uncoupled
mitochondria respiring on succinate, there is an increased production of oxaloacetate [35], a potent inhibitor of complex II activity.
The rate of removal of oxaloacetate dictates the rate of complex
II reactivation [39]. Accumulation of oxaloacetate, and subsequent
inhibition of succinate oxidase activity, is prevented by addition of
rotenone [3537]. Oxaloacetate production is impeded by rotenone because of the large [NADH]/[NAD+] ratio that is maintained
during state 3 respiration, which in turn prevents the forward malate dehydrogenase reaction. Thus under conditions in which oxaloacetate production is halted, H2O2 has minimal effect on complex
II activity.
Our results demonstrate that H2O2 is preventing the reactivation of complex II when transitioning from state 3 to state 4 respiration because of either enhanced production or decreased
capacity to clear oxaloacetate. However, it was found that MDH
is unaffected by H2O2 under our experimental conditions. The
accumulation of oxaloacetate is therefore likely due to inhibition
of other Krebs cycle enzymes, such as aconitase [19], that inuence
oxaloacetate production and consumption. Alternatively, the oxaloacetate that is produced at an elevated rate during state 3 may
undergo a direct, nonenzymatic, reaction with H2O2 [4143]. The
product of this decarboxylation reaction is malonate, a classical
inhibitor of complex II. Thus, multiple mechanisms involving oxaloacetate may contribute to H2O2-mediated inhibition of succinate-linked respiration.
The complex II assay employed in this study directly measured
the reduction of an ubiquinone analog (ubiquinone-1) [27]. This
assay is a function of both succinate oxidation and electron transfer
to ubiquinone and is TTFA sensitive. The traditional assay of succinate dehydrogenase (SDH) activity measures the phenazine
methosulfate (PMS) mediated reduction of 2,6-dichloroindophenol
(DCIP) [44]. This reaction is insensitive to TTFA, as transfer of electrons to ubiquinone is not required for DCIP reduction, but is sensitive to inhibition by oxaloacetate. It was determined that SDH
activity, as measured by PMS/DCIP assay, followed the same pattern of activity. That is, activity in control samples dropped as a
function of state 3 respiration and recovered in state 4. Importantly, this activity was also sensitive to H2O2 and showed the
same slower rate of recovery when transitioning to state 4. This result suggests loss of enzyme activity occurs at the level of succinate
oxidation and supports the supposition that H2O2 prevents SDH
reactivation, at least in part, by maintaining inhibitory levels of
oxaloacetate.
Previous studies have examined the susceptibility of complex II
to H2O2-mediated inactivation with varying results. Complex II is
not inhibited by H2O2 in submitochondrial particles [20], but has
been reported to be inhibited in intact mitochondria [18,19]. In
cardiac mitochondria respiring on a-ketoglutarate, complex II
activity is reversibly inhibited by H2O2 during state 3 respiration
[19]. That study, however, did not consider the dynamic regulation
of complex II when transitioning from state 3 to state 4 respiration.
The respiratory substrate available is also likely to affect susceptibility of complex II to oxidative inactivation. For example, complex
II has been shown to be sensitive to inhibition by the highly
reactive lipid peroxidation product, 4-hydroxy-2-nonenal, in mitochondria respiring in the presence of succinate but not a-ketoglutarate [32,40]. Future studies need to consider not only the
susceptibility of complex II to oxidative inactivation as a function
of metabolic state, but also the available energy source.
While it is well known that oxaloacetate is a potent inhibitor of
complex II activity, the physiological relevance of this inhibition
remains obscure. Oxaloacetate is a transient intermediate of the
Krebs cycle that does not accumulate in readily measurable
amounts in mitochondria. However, the results of the present work

M.D. Moser et al. / Archives of Biochemistry and Biophysics 488 (2009) 6975

demonstrate oxaloacetate-mediated inhibition of complex II can be

modulated as by micromolar concentrations of H2O2. This increase
may be signicant under certain physiological conditions in which
H2O2 production is increased, and inhibition of complex II may be
desirable. For example, ischemic preconditioning is accompanied
by increased mitochondrial free radical production (reviewed in
[45]), which likely acts as a trigger for pro-survival signaling pathways (reviewed in [46]). Interestingly, pharmacological reagents
that inhibit complex II activity can mimic the benecial effects of
ischemic preconditioning [4750]. However, the importance of
oxaloacetate in inhibiting complex II during ischemic preconditioning is currently unknown. This work provides new insight into
how certain physiological conditions of increased free radical production may inuence complex II activity via increased oxaloacetate production.
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