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Archives of Biochemistry and Biophysics 488 (2009) 69–75

Archives of Biochemistry and Biophysics 488 (2009) 69–75 Contents lists available at ScienceDirect Archives of

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

journal homepage: www.elsevier.com/locate/yabbi Inhibition of succinate-linked respiration and complex II

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

article info

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

abstract

Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxi- dant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological con- centrations. Previous studies, however, have suggested that complex II (succinate dehydrogenase) is sensitive to H 2 O 2 -mediated inhibition. Nevertheless, the effects of H 2 O 2 on succinate-linked respiration and complex II activity have not been examined in intact mitochondria. Results presented indicate that H 2 O 2 inhibits succinate-linked state 3 mitochondrial respiration in a concentration dependent manner. H 2 O 2 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 H 2 O 2 on succinate-linked respiration and complex II activity. It is concluded that H 2 O 2 inhibits succinate-linked respiration indirectly by sus- taining and enhancing oxaloacetate-mediated inactivation of complex II. 2009 Elsevier Inc. All rights reserved.

Introduction

Complex II (succinate dehydrogenase, succinate–ubiquinone oxidoreductase) is a multifunctional enzyme, catalyzing the oxida- tion of succinate to fumarate as part of the Krebs cycle and concur- rently 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 flow [1,2] . Reverse electron flow is also responsible, in part, for the production of reac- tive oxygen species produced in mitochondria respiring on succi- nate as an energy source [3–9] . Thus, regulation of complex II activity is critical to both modulate its activity in response to ener- getic 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 intermedi- ates. 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 po- tent inhibitor of complex II activity [11–13]. 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 inacti- vated 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, oc- curs as a function of oxaloacetate removal and production, ubiqui- none 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 com- plex 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 H 2 O 2 , under normal conditions and increased amounts under con- ditions of oxidative stress. H 2 O 2 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 H 2 O 2 [18–20]. However, enzymes that contain redox sensitive prosthetic groups or reactive thiols in their active sites display sensitivity to low concentrations of H 2 O 2 either by di- rect oxidation or via the formation of protein–glutathione mixed disulfides [21] . It has previously been shown that complex II is one of select few mitochondrial enzymes that is inhibited by H 2 O 2 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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by which H 2 O 2 inhibits complex II have not been examined. The goals of the present study were to therefore to define the mecha- nism(s) H 2 O 2 -mediated complex II inhibition, specifically as a function of the mitochondrial metabolic status, and determine how H 2 O 2 affects succinate-linked respiration.

Materials and methods

Isolation of cardiac mitochondria

Sprague–Dawley rats were anesthetized with a mixture of ket- amine, xylazine, and acepromazine (3:3:1) (0.5–0.75 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 500 g for 5 min at 4 C, the superna-

tant collected and passed through cheese cloth, and spun again for

10 min at 5000 g . The resulting mitochondrial pellet was resus-

pended in approximately 200 l L of buffer A and protein concentra- tion 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 KH 2 PO 4 , and 10 mM MOPS, pH 7.4 (buffer

B) containing 10 mM succinate. Respiration was measured polaro- graphically at 25 C with a Clark-style oxygen electrode (Instech) in the presence or absence of 1.0 l M rotenone and indicated concen- trations of H 2 O 2 . State 2 respiration is defined 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 atmo- spheric pressure and 20 C. A high concentration of succinate (10 mM) was necessary to achieve maximal rates of state 3 respi- ration, particularly when oxygen consumption was measured in the absence of rotenone.

H 2 O 2 consumption

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

10 mM succinate, 1.0 l M rotenone, and H 2 O 2 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 K 2 HPO 4 , pH 7.25 with 0.1% Triton X-100, 0.5 mM hydroxyphenylacetic acid and 2.0 U/mL horseradish per- oxidase. H 2 O 2 was measured fluorometrically using 320 nm excita- tion/425 nm emission [25,26].

Enzyme activities

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

10 mM succinate, 1.0 l M rotenone, and H 2 O 2 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 contain- ing 25 mM MOPS pH 7.8, 5.0 mM MgCl 2 , 2.0 l g/mL antimycin A, and 0.1% Triton X-100. It was found that this detergent solubiliza- tion methodology effectively locked complex II into either the ac- tive or inactive state that was present in the intact mitochondria. Complex II activity was measured spectrophotometrically by the

thenoyltrifluoroacetone (TTFA) sensitive reduction of ubiquinone-

1 (50 l M) 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 final 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 indi- cated 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 + .

NAD(P)H measurements

Mitochondria were diluted to 0.25 mg/mL in buffer B with 10 mM succinate, 1.0 l M rotenone, and H 2 O 2 as indicated. NAD(P)H autofluorescence was measured using 340 nm excita- tion/460 nm emission [29] . NADH and NADPH autofluorescence 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 H 2 O 2 as indicated. State 3 respiration was initiated by addition of 0.5 mM ADP at 2.0 min, and rotenone (1.0 l M) 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 14,000 g ), and the supernatant ana- lyzed by isocratic reverse-phase HPLC using with a mobile phase of pH 5.0 10 mM KH 2 PO 4 at a flow rate of 1.0 mL/min. NADH and NADPH were monitored by fluorescence detection (340 nm excita- tion/460 nm emission) and identification verified by comparison of peak retention times to NADH and NADPH standards.

Statistical analysis

Data are presented as means ± SE. The data were evaluated uti- lizing a two-tailed t -test. Statistical significance was assigned for p 6 0.05, as indicated.

Results

The effects of H 2 O 2 on succinate-linked respiration

To determine the effect of H 2 O 2 on succinate-linked respiration, cardiac mitochondria were isolated and respiration measured in the presence or absence of H 2 O 2 . As shown in Fig. 1 A, control car- diac 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 [11–13], while the increase in the third phase is likely mediated

by reactivation of complex II by ATP [12,15]. H 2 O 2 has no effect on the fast, initial rate of state 3 respiration, but inhibits phase 2 state

3 respiration in a concentration dependent manner. H 2 O 2 (50 l M)

inhibits the rate of state 3 respiration by 40%, but has no significant

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) 69–75

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of Biochemistry and Biophysics 488 (2009) 69–75 71 Fig. 1. Hydrogen peroxide inhibits succinate-linked state 3

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 H 2 O 2 was present, it was added at t = 0. (A) Mitochondria were incubated with 0 (black), 25 l M (blue), or 50 l M (red) H 2 O 2 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 l M rotenone in the absence (solid) or presence (hatched) of 50 l M H 2 O 2 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 H 2 O 2 . Rat heart mitochondria (0.25 mg/mL) were incubated at 25 C with 10 mM succinate in the presence or absence of 50 l M H 2 O 2 and/or 1.0 l M 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 O 2 min 1 mg 1 ± SE (n P 5 for each experimental condition).

 

State 3

State 4

RCR

Control

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

H 2 O 2 Rotenone Rotenone + H 2 O 2

50 l M H 2 O 2 (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 l M rotenone, a concentration of complex I inhibitor that was found to completely block NADH-linked respiration. While rote- none can increase superoxide production during NADH-linked res- piration [30] , addition of the inhibitor to succinate-linked respiration blocks superoxide production, and thus hydrogen per- oxide production, by preventing reverse electron flow [8,31]. Thus endogenous H 2 O 2 production was not a confounding factor under

these experimental conditions. Under control conditions, rotenone increased state 3 respiration by 69% and doubled the RCR as com- pared to respiration in its absence ( Fig. 1 B and Table 1 ). Interest- ingly, H 2 O 2 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 H 2 O 2 clearance or antioxidant capacity. The rate of consumption of 50 l M H 2 O 2 by mitochondria, in the presence or absence of rotenone, was found to be identical and essentially complete by 15 min (11.2 nmol H 2 O 2 min 1 mg 1 for mitochondria with succinate alone and 10.4 nmol H 2 O 2 min 1 mg 1 for mitochondria with succinate and rotenone).

Reversibility of H 2 O 2 -mediated inhibition and protection by ATP

To demonstrate the reversibility of H 2 O 2 -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 H 2 O 2 results in an immediate up- ward deflection in O 2 concentration, indicating the rapid decompo- sition of H 2 O 2 to H 2 O and O 2 . Importantly, this removal of H 2 O 2 completely prevented deficits in state 3 respiration. The transient inhibition of H 2 O 2 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. 3 A) in control mito- chondria. Importantly, reinitiating state 3 respiration resulted in the same accelerated rate in both control and H 2 O 2 -treated mito- chondria ( Fig. 3 A), thus demonstrating the effects of H 2 O 2 on respi- ration are reversible. Furthermore, mitochondria in state 4 (ATP present) were found to be resistant to further decreases in respira- tion upon addition of another bolus of 50 l M H 2 O 2 prior to second addition of ADP (data not shown). The reason for this, it was pos- tulated, was that ATP present in state 4 acted to protect against H 2 O 2 -mediated inhibition. This was confirmed by the experiment in Fig. 3 B, which demonstrates addition of ATP prior to state 3 in- creased the maximal rate of respiration and completely prevented H 2 O 2 -mediated inhibition.

Complex II activity and H 2 O 2

It has been previously reported that complex II is sensitive to oxidative inactivation [19,32]. We therefore examined whether H 2 O 2 -mediated inhibition of succinate-linked respiration was

-mediated inhibition of succinate-linked respiration was Fig. 2. H 2 O 2 -induced inhibition of

Fig. 2. H 2 O 2 -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 l M H 2 O 2 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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/ Archives of Biochemistry and Biophysics 488 (2009) 69–75 Fig. 3. H 2 O 2 -induced

Fig. 3. H 2 O 2 -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 l M H 2 O 2 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 l M H 2 O 2 , 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 find- ings, 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 activa- tion was unaffected by 50 l M H 2 O 2 . Both control and H 2 O 2 -treated samples showed a dramatic decrease in complex II activity upon addition of ADP, a phenomenon attributable to the increase in oxa- loacetate production during state 3 respiration [11–13] . Notewor- thy, the H 2 O 2 -treated sample demonstrated an enhanced inactivation of complex II activity during state 3 respiration ( Fig. 4 A), 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 corre- sponded to the transition to state 4 respiration. Complex II activity in H 2 O 2 -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 respir- ing on succinate in the presence of rotenone ( Fig. 4 B). In control mitochondria, rotenone largely prevented the inactivation of com- plex 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 reac- tion catalyzed by malate dehydrogenase (MDH) to produce oxalo-

catalyzed by malate dehydrogenase (MDH) to produce oxalo- Fig. 4. Complex II activity is decreased during

Fig. 4. Complex II activity is decreased during state 3 respiration and this is exacerbated by H 2 O 2 , but prevented by rotenone. Mitochondria (0.25 mg/mL) were incubated in the absence (dark bars) or presence (light bars) of 50 l M H 2 O 2 , 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 l L 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) Mito- chondria were incubated in the presence of 10 mM succinate alone. Symbols ( ) indicate statistical significance ( 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 l M rotenone. Values are means ± SE ( n P 5 for each experimental condi- tion). (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 H 2 O 2 ( Fig. 4 B). 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) 69–75

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H 2 O 2 -treated mitochondria, indicating that a slight inactivation of complex II is not sufficient to impede the maximal rate of succi- nate-linked respiration. The effect of ATP on complex II activity was next examined. ATP, which prevented H 2 O 2 -induced loss of state 3 respiration ( Fig 3 B), also largely prevented inactivation of complex II in control mitochondria and H 2 O 2 -treated mitochondria during state 3 respiration. Upon initiation of state 3 respiration in the presence of ATP, complex II activity decreased only 17% in con- trol mitochondria and 27% in H 2 O 2 -treated mitochondria as com- pared 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 ef- fect ATP has on MDH activity is demonstrated in Fig. 4 C in which activity was assayed in the presence of increasing concentrations of ATP. As the figure illustrates, the rate of oxaloacetate formation followed first 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 H 2 O 2 -mediated complex II inactivation

Several lines of evidence suggest H 2 O 2 is inhibiting succinate- linked respiration by a mechanism that induces increased inactiva- tion of complex II activity during state 3 respiration, and not via di- rect oxidation of the enzyme. For example, complex II activity is completely resistant to H 2 O 2 -mediated inhibition during state 2 respiration. In addition, both rotenone and ATP completely blocked H 2 O 2 -mediated inhibition of state 3 respiration and largely pre- vented H 2 O 2 -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. Rote- none prevents the accumulation of oxaloacetate during state 3 res- piration by maintaining a large ratio of [NADH]/[NAD + ] [35–37], thereby sustaining complex II in the active state. This suggests that the effect of H 2 O 2 on succinate-linked respiration is to enhance the accumulation of oxaloacetate, thereby leading to depressed com- plex II activity. However, a direct assessment of oxaloacetate con- centrations 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 oxa- loacetate involvement can be tested. Oxaloacetate, when gluta- mate 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 l M H 2 O 2 . A low concentration of gluta- mate (1.0 mM) was used, which on its own, was unable to support state 3 respiration ( Fig. 5 A). However, this concentration of gluta- mate enhanced succinate-linked state 3 and prevented the second, slow phase, of state 3 respiration that is normally present in con- trol mitochondria ( Fig. 5 A). Importantly, glutamate completely prevented H 2 O 2 -mediated inhibition of state 3 respiration ( Fig. 5 A). Furthermore, it was found that glutamate prevents inac- tivation of complex II activity during state 3 respiration in control mitochondria and attenuated the effect of H 2 O 2 ( Fig. 5 B).

Modulation of NAD(P)H by H 2 O 2

Our results suggest H 2 O 2 is inhibiting succinate-linked respira- tion 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

]/[NADH] and [malate]/[oxaloacetate] [39] . The status of Fig. 5. Glutamate prevents H 2 O 2

Fig. 5. Glutamate prevents H 2 O 2 -mediated inhibition of succinate-linked respira- tion 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 l M H 2 O 2 (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 l M H 2 O 2 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 autofluorescence, can therefore provide insight into conditions that promote oxaloacetate produc- tion. As shown in Fig. 6 A, 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 produc- tion. Treatment of mitochondria with H 2 O 2 (50 l M) did not affect the magnitude of NAD(P)H oxidation, but did extend the duration of oxidative conditions. These extended oxidative conditions corre- spond 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 flow [2] . Thus, the increase in NAD(P)H upon addi- tion of rotenone ( Fig. 6 B) during state 3 respiration is mediated by the reduction of NAD + by Krebs cycle dehydrogenases and preven- tion of consumption of NADH by complex I. In the presence of H 2 O 2 , 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 H 2 O 2 . Removal of H 2 O 2 by catalase restored NAD(P)H levels to control

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/ Archives of Biochemistry and Biophysics 488 (2009) 69–75 Fig. 6. H 2 O 2 induces

Fig. 6. H 2 O 2 induces rotenone-insensitive consumption of NAD(P)H in mitochon- dria respiring on succinate. Mitochondria (0.25 mg/mL) were incubated in the absence (black traces) or presence of 50 l M H 2 O 2 (red traces) as in Fig. 1 . NAD(P)H autofluorescence was measured using 340 nm excitation/460 nm emission [29] . NADH and NADPH autofluorescence are indistinguishable, and therefore measure- ments 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 l M) was added at 4.0 min while mitochondria were still in state 3 respiration. Catalase (200 U) was added at 10 min.

conditions ( Fig. 6 B). This consumption of NAD(P)H is likely attrib- utable to malate dehydrogenase catalyzing the consumption of oxaloacetate to malate. The fluorescence measurements in Fig. 6 represent the sum of autofluorescence contributions from NADH and NADPH, although the concentration of NADH is much higher than NADPH in mito- chondria [29,40]. Nevertheless, it may be expected that H 2 O 2 -dri- ven decrease in NAD(P)H fluorescence is attributable to the activities of glutathione reductase or thioredoxin reductase which consume NADPH as part of the mitochondrial glutathione peroxi- dase and peroxiredoxin 3 H 2 O 2 detoxification systems, respec- tively. 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 H 2 O 2 -driven decrease in NAD(P)H autofluorescence seen in Fig. 6 B 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 transhydrogen- ase reaction that replenishes NADPH at the expense of NADH.

Discussion

The present report demonstrates that H 2 O 2 inhibits succinate- linked 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 produc- tion 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 [35–37]. Oxaloacetate production is impeded by rote- none because of the large [NADH]/[NAD + ] ratio that is maintained during state 3 respiration, which in turn prevents the forward ma- late dehydrogenase reaction. Thus under conditions in which oxa- loacetate production is halted, H 2 O 2 has minimal effect on complex

II activity.

Our results demonstrate that H 2 O 2 is preventing the reactiva- tion of complex II when transitioning from state 3 to state 4 respi-

ration because of either enhanced production or decreased capacity to clear oxaloacetate. However, it was found that MDH

is unaffected by H 2 O 2 under our experimental conditions. The

accumulation of oxaloacetate is therefore likely due to inhibition

of other Krebs cycle enzymes, such as aconitase [19] , that influence

oxaloacetate production and consumption. Alternatively, the oxa- loacetate that is produced at an elevated rate during state 3 may undergo a direct, nonenzymatic, reaction with H 2 O 2 [41–43]. The product of this decarboxylation reaction is malonate, a classical inhibitor of complex II. Thus, multiple mechanisms involving oxa- loacetate may contribute to H 2 O 2 -mediated inhibition of succi- nate-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 succi-

nate 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 elec- trons to ubiquinone is not required for DCIP reduction, but is sen-

sitive to inhibition by oxaloacetate. It was determined that SDH activity, as measured by PMS/DCIP assay, followed the same pat- tern of activity. That is, activity in control samples dropped as a function of state 3 respiration and recovered in state 4. Impor- tantly, this activity was also sensitive to H 2 O 2 and showed the same slower rate of recovery when transitioning to state 4. This re-

sult suggests loss of enzyme activity occurs at the level of succinate oxidation and supports the supposition that H 2 O 2 prevents SDH reactivation, at least in part, by maintaining inhibitory levels of oxaloacetate. Previous studies have examined the susceptibility of complex II

to H 2 O 2 -mediated inactivation with varying results. Complex II is

not inhibited by H 2 O 2 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 H 2 O 2 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 suscepti-

bility 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 mito- chondria respiring in the presence of succinate but not a -ketoglu- tarate [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) 69–75

75

demonstrate oxaloacetate-mediated inhibition of complex II can be modulated as by micromolar concentrations of H 2 O 2 . This increase may be significant under certain physiological conditions in which H 2 O 2 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 path- ways (reviewed in [46] ). Interestingly, pharmacological reagents that inhibit complex II activity can mimic the beneficial effects of ischemic preconditioning [47–50]. However, the importance of oxaloacetate in inhibiting complex II during ischemic precondi- tioning is currently unknown. This work provides new insight into how certain physiological conditions of increased free radical pro- duction may influence complex II activity via increased oxaloace- tate production.

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