relative contributions of heart mitochondria glutathione peroxidase and catalase to h2o2...

Original Contribution

RELATIVE CONTRIBUTIONS OF HEART MITOCHONDRIA GLUTATHIONEPEROXIDASE AND CATALASE TO H2O2 DETOXIFICATION IN

IN VIVO CONDITIONS

FERNANDO ANTUNES,*† DERICK HAN,* and ENRIQUE CADENAS**Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles,CA, USA; and†Centro de Estudos de Bioquı´mica e Fisiologia and Grupo de Bioquı´mica e Biologia Teo´ricas (Instituto Bento da

Rocha Cabral), Universidade de Lisboa, Lisboa, Portugal

(Received 30 April 2002;Revised 25 June 2002;Accepted 11 July 2002)

Abstract—This study was aimed at assessing the relative contributions to H2O2 detoxification by glutathione peroxidaseand catalase in the mitochondrial matrix of heart. For this purpose, mitoplasts from rat heart were used in order tominimize contamination with microperoxisomes, and the kinetic rate constants of both enzymatic activities weredetermined along with a simulation profile. Results show that the contribution of catalase to H2O2 removal in heartmitochondria is not significant, even under strong oxidative conditions, such as those achieved in ischemia-reperfusionand involving extensive glutathione depletion and high H2O2 concentrations. Conversely, maintenance of the steadystate levels of H2O2 in the heart mitochondrial matrix seems to be the domain of glutathione peroxidase. It is suggestedthat the physiological role of the low amounts of catalase found in heart mitochondria is related to its peroxidatic ratherthan catalatic activity. © 2002 Elsevier Science Inc.

Keywords—Hydrogen peroxide, Catalase, Glutathione peroxidase, Nitric oxide, Glutathione, Simulation, Free radicals

INTRODUCTION

Mitochondria are key cellular sites for the production ofhydrogen peroxide (H2O2) [1], an oxidant that can rep-resent a hazard to the cell at high concentrations, but thatalso has a regulatory role in several processes, includingsignal transduction, development, cell proliferation, andapoptosis [2]. Mitochondrial density is high in heart,constituting up to 35% of the cell volume of rat heartmuscle cells [3] and, expectedly, oxidative stress plays akey role in several cardiac disorders, such has ischemia-reperfusion, inflammation, myocardial infarction, and ar-rhythmias [4].

The detoxification of H2O2 and the consequent con-trol of its steady state concentration are, therefore, of keyrelevance: glutathione-dependent peroxidases and cata-lase are the main enzymes responsible for the cellularremoval of H2O2. The classic glutathione peroxidase(GPx, EC 1.11.1.9) is the most important peroxidase for

H2O2 removal in mammals [5,6]. The enzyme is presentin cytosol, mitochondria, endoplasmic reticulum, andnuclei [7,8], whereas catalase (EC 1.11.1.6) is present inperoxisomes and at low levels in the cytosol [9,10] and inheart mitochondria [11]. In general glutathione peroxi-dases are more important than catalase removing H2O2

[12,13], but catalase has a predominant role at least inperoxisomes where it is concentrated [1]. In heart mito-chondria, catalase comprises 0.025% of the protein [11]and its activity prevents damage induced by H2O2 atconcentration of this species that overcome the glutathi-one system [14], but the relative contributions of catalaseand glutathione peroxidase under physiological condi-tions are unknown.

The relative contribution of glutathione peroxidaseand catalase in the removal of H2O2 gains further sig-nificance when considering that detoxification via cata-lase does not represent a stress to the cell (as H2O2 isconverted to O2 and H2O), whereas that via glutathioneperoxidase is accomplished with the concomitant oxida-tion of glutathione (GSH), thus imposing both an oxida-tive- and an energetic stress to the cell.

Address correspondence to: Fernando Antunes, Centro de Estudos deBioquımica e Fisiologia, Universidade de Lisboa, Ed. C8, CampoGrande, 1749-016 Lisboa, Portugal; Tel:�351 (21) 750-0916; Fax:�351 (21) 750-0961; E-Mail: [email protected].

Free Radical Biology & Medicine, Vol. 33, No. 9, pp. 1260–1267, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/02/$–see front matter

PII S0891-5849(02)01016-X

1260

This study was aimed at providing a rigorous quanti-tative analysis of the contributions of heart mitochondriacatalase and glutathione peroxidase to the detoxificationof H2O2 under conditions that mimic in vivo situations.

MATERIALS AND METHODS

Chemicals, biochemicals, and biological materials

Digitonin and glutathione reductase (Baker’s yeast)were from Fluka (Buchs, Switzerland). Nicotinamideadenine dinucleotide phosphate (NADPH) (98%) wasfrom Boehringer (Mannheim, Germany). Diethylene-treaminepentaacetic acid (DTPA), GSH, GSSG, H2O2,NaN3, and Triton X-100 were from Sigma Chemical Co.(St. Louis, MO, USA). All other chemicals were ofanalytical grade.

Mitoplasts were prepared from heart mitochondriaisolated from adult male Wistar rats by differential cen-trifugation in an isolation buffer consisting of 230 mMmannitol, 70 mM sucrose, 1 mM ethylenediaminetet-raacetic acid (EDTA), and 5 mM Trizma/HCl buffer, pH7.4 [15]. Mitoplasts were generated by hypo-osmotictreatment [16]: isolated rat heart mitochondria were sus-pended in cold hypotonic medium [10 mM KCl and 2mM N-[2-hyroxyethyl]piperazine-N'-[2-ethanesulfonicacid]) (HEPES), pH 7.4] for 20 min at a concentration of1 mg/ml [16]. The suspension was centrifuged at 8000 �g for 10 min, and resuspended in a solution containing150 mM KCl and 2 mM HEPES, pH 7.2. The resus-pended pellet was centrifuged at 8000 � g and redis-solved in 150 mM KCl. After a final centrifugation, thepellet was dissolved in isolation buffer described aboveat a concentration of 8 mg/ml [17].

ENZYMATIC ACTIVITIES

Catalase activity was measured as previously de-scribed [18] in isolation buffer with 0.01% digitonin.Digitonin was used to lyse mitoplasts, for enzyme activ-ity in intact mitoplasts was below the detection limit.H2O2 (10 mM initial concentration) consumption wasfollowed at 240 nm at room temperature for 2 minagainst a blank without H2O2. Glutathione peroxidaseactivity was measured by assessing whole cell kineticsaccording to methods previously described [19,20].Briefly, oxidation of glutathione by H2O2 catalyzed byGPx is coupled to the reduction of glutathione byNADPH catalyzed by an excess of glutathione reductase.Concentrations of H2O2 during the assay were estimatedfrom NADPH oxidation as described in [20], and werefitted to Eqn. 2.

RESULTS AND DISCUSSION

Characterization of the kinetic rate constants for cata-lase and glutathione peroxidase is a requisite condition todetermine the relative contribution of these enzymes toH2O2 removal [19] in heart mitochondria. Rat heartmitoplasts were used to perform this analysis in order tominimize contamination with microperoxisomes [11].The functional integrity of mitochondria and mitoplastswas assessed in terms of the respiratory control ratio(defined as the state 3/state 4 ratio). Mitoplasts remainedfunctionally similar to mitochondria: The respiratorycontrol ratios in presence of glutamate/malate with intactmitochondria were 5.0 � 0.9; those obtained with mito-plasts were 4.2 � 1.1. This slight decrease is usuallyobserved with mitoplasts [17].

Catalase kinetics

Catalase does not show saturation for H2O2 [18] and,therefore, the consumption of H2O2 by catalase follows afirst order kinetics (Fig. 1). The slope of the plot in Fig.1 represents a pseudo-first order rate constant (kcat),which reflects the capacity of the enzyme to removeH2O2. Catalase activity obtained with lysed mitoplastswas 1.5 � 0.5 � 10�2 s�1 (mg of protein)�1 (n � 3). Inintact mitoplasts, i.e., without digitonin treatment, cata-lase activity was below the detection limit of the method.

Glutathione peroxidase activity

The mechanism of reaction of glutathione peroxidaseinvolves an oxidation-reduction cycle of the Se-cysteine

Fig. 1. Catalase activity in isolated heart mitoplasts. Catalase activitywas measured in the presence of digitonin as described in the Materialsand Methods section. The reaction mixture consisted of digitonin0.01% (in dimethyl sulfoxide [DMSO]) plus mitoplasts (0.05 mg) and10 mM H2O2.

1261Glutathione peroxidase and catalase in heart mitochondria

residue at the active center using GSH as the reducingagent [21]:

H2O2 � GPxred � H�O¡

kGPxrd

GPxox � H2O (1)

GPxox � GSHO¡k2

GS-GPx � H2O (2)

GS-GPx � GSHO¡k3

GPxred � GSSG � H� (3)

Extensive work from Flohe’s and Tappel’s laborato-ries [20,22] found that the kinetics of the enzyme fol-lowed a ping-pong mechanism as described by Eqn. 1.This equation is derived assuming the steady state ap-proximation for the different GPx forms (� refers to therate of H2O2 oxidation, [GPxtot] refers to total concen-tration of glutathione peroxidase, �1 � 1/kGPxred, and �2

� 1/k2 � 1/k3).

�GPxtot�

��

�1

�H2O2��

�2

�GSH�(1)

In the coupled assay used in this work, GSH is keptconstant because of its reduction by glutathione reduc-tase; therefore, integration of Eqn. 1 yields Eqn. 2, where[H2O2]t and [H2O2]0 refer to the concentration of H2O2

in the assay at time t and initially, respectively.

�GPxtot � t�

�H2O2�0 � �H2O2� t� �2/�GSH� � �1 �

ln([H2O2]0/�H2O2� t)

�H2O2]0 � �H2O2� t

(2)

Equation 2 has been used to study not only purifiedglutathione peroxidase [20,23], but also glutathione per-oxidase in whole homogenates of Jurkat-T cells [19]. Asshown in Fig. 2, glutathione peroxidase kinetics in mi-toplasts fitted well Eqn. 2. The following parameterswere obtained: [GPxtot]/�1 � kGpxred

[GPxtot] � (6.4 �0.4) � 10�1 s�1 (mg of protein)�1 (n � 7) (Fig. 2A) and[GPxtot]/�2 � (1.33 � 0.03) � 10�3 s�1 (mg of pro-tein)�1 (n � 7) (Fig. 2B).

The kinetic method used does not allow determinationof �1 and �2 without the knowledge of [GPxtot] but thevalue of �1/�2 ratio, which reflects the redox propertiesof glutathione peroxidase, was determined and compareswell with those obtained with Jurkat T-cells [19], ratliver [20], and bovine erythrocytes [21] (Table 1).

Relative contribution of GPx and catalase in vivo

The terms kGpxred[GPx]tot [H2O2] and kcat [H2O2]

characterize the consumption of H2O2 by glutathioneperoxidase at low H2O2 concentrations [19] and catalase,respectively. Hence, the relative contribution of catalaseand glutathione peroxidase to the removal of H2O2 underthe low levels of H2O2 found in vivo is given from theratio kGPxrd

[GPxtot]/kcat. The value obtained was 44 � 9(n � 3) and, therefore, under low H2O2 levels and whenthe glutathione system is not overload, catalase contri-bution for H2O2 removal represents, at most, 2.3% of thetotal. This value may be considered an upper limit be-cause it is assumed that: (i) there is not a peroxidaticreaction (i.e., oxidation of small compounds) competingwith the catalatic (i.e., H2O2 removal) activity of cata-lase; and (ii) all catalase activity measured reflects truemitochondrial catalase and not minor contaminationfrom peroxisomes.

GSH levels and the activities of glutathione peroxidaseand catalase

Conditions of oxidative stress, such as GSH depletionand increase of H2O2 steady state levels, may favor thecatalase pathway for H2O2 removal. This notion wasaddressed by a derived equation that relates the pathwaysof H2O2 elimination with the concentrations of GSH andH2O2. Based on Eqn. 1 and assuming that the rate ofH2O2 removal by catalase is given by �cat � kcat [H2O2],Eqn. 3 is derived; this equation provides the ratio be-tween the H2O2 removed via GPx and via catalase.

�GPx

�cat�

�GPxtot�

�1

�H2O2��

�2

�GSH�

kcat�H2O2�(3)

After algebraic manipulation, this equation can berewritten as follows:

�GPx

�cat�

�GPxtot�

�1kcat

1

1

�H2O2��

1

�GSH�

�2

�1

�H2O2�(4)

Table 1. �1/�2 Values for Glutathione Peroxidase Activity fromDifferent Sources

�1/�2 Reference

Rat heart mitoplasts 4.8 � 102 This workJurkat T-cells 4.7 � 102 [19]Rat liver 5.3 � 102 [20]Bovine erythrocytes 1.2 � 102 [21]

1262 F. ANTUNES et al.

Equation 4 is advantageous because instead of fourunknowns—[GPxtot], �1, �2, and kcat—there are onlytwo unknowns: two ratios—[GPxtot]/(�1kcat), and �1/�2. These ratios were obtained experimentally and,therefore, errors originating from conversion of unitsfrom the experiment in vitro to the situation in vivo areabrogated.

By changing the concentrations of GSH and H2O2, therelative contributions of catalase and glutathione perox-

idase under oxidative stress in vivo can be simulated(Fig. 3). For the physiological range of H2O2 concentra-tions (0.01–0.1 �M [1]), catalase contribution was 2.3%even with conditions implying 99% of GSH depletion. Ifa content of GSH in heart mitochondria of around 8 mMis assumed, 99% GSH depletion causes the concentrationof glutathione to drop to 0.08 mM, a value higher thanthe H2O2 concentration assumed (0.1 �M); under theseconditions, glutathione peroxidase is still saturated with

Fig. 2. Kinetics of glutathione peroxidase activity in isolated mitoplasts. (A) Kinetic analysis was performed as described in theMaterial and Methods section; results were fitted to the integrated rate law of glutathione peroxidase. (B) The intercepts obtained in(A) were plotted as a function of the reciprocal of GSH concentration ([GSH]�1). Measurements were made as follows: mitoplast (0.05mg of protein/ml), 0.05 M potassium phosphate buffer pH 7.0, 1 mM DTPA, 50 mM sodium azide, 1.1 U/ml glutathione reductase,0.1 mM NADPH, 35 mM H2O2, GSH (0.66–3.0 mM), and 1% Triton X-100 (v/v). Reaction was initiated by the addition of H2O2 aftera preincubation of 10 min, and NADPH oxidation was followed at 340 nm.


GSH and fully operational. For a concentration of H2O2

of 1 �M, a level that is sufficient to cause apoptosis inJurkat T-cells [24], the contribution of catalase would be3.5% and 14%, corresponding to a 90% and 99% deple-tion of GSH, respectively.

In this regard, it is worth noting that: (i) cells havesome tolerance for a temporary large (90%) depletion ofcytosolic GSH, but not of mitochondrial GSH [25] and(ii) in ischemia-reperfusion, a situation of acute oxidativestress generated in vivo with induction of apoptosis andnecrosis [26], the levels of GSH depletion are typicalaround 50% [27], a relative low value that may be theresult of the slow half-life of GSH in the heart (69.6 h)[28]. Therefore, the 90% of GSH depletion window(range) shown in Fig. 3 probably covers the depletionsthat may be achieved in vivo in the heart. This analysiscan also be applied to evaluate the impact of decreasedavailability of NADPH: NADPH is fundamental to keepGSH levels in mitochondria [29], and any impairment inthe mitochondrial pathway of NADPH production,which includes a transhydrogenase pathway [25], wouldaffect H2O2 removal by decreasing the availability ofGSH.

If the concentration of H2O2 were 10 �M (i.e., 100-fold higher than the upper limit for the physiologicallevel) a GSH depletion of 90% would suggest a contri-bution of catalase of 14%. In this regard, it is worthnoticing that the maximal increase observed for H2O2 inischemia-reperfusion is approximately 6-fold [30]. Inorder to observe a significant contribution of catalase toH2O2 removal, extreme oxidative conditions need beassumed: high H2O2 concentration [31] and almost com-

plete GSH depletions. Such conditions may be achievedin vitro [14] but not in vivo.

Any conditions that change the activity of catalaseand GPx affect this analysis. For example, reactive ox-ygen species, including superoxide anion, H2O2, and•NO, damage catalase [32–34] and GPx [35–37], thusdecreasing their activity. On the other hand, oxidantsinduce adaptive changes in the cell, and gene expressionof these enzymes is activated, thus increasing their ac-tivity [38]. The predominant effect is dependent of theconcentration of the oxidant: small increases over thephysiological concentration are expected to induce adap-tive changes while damaging actions are expected athigher concentrations. Overall, a rigorous analysis ofthese effects will require a titration of cellular responseswith the oxidant under experimental conditions thatmimic the state in vivo.

Taking into account the marked difference in activitiesfor catalase and GPx found in this work, it may be surmisedthat under the oxidative conditions attainable in vivo thecontribution of catalase to detoxification of H2O2 in heartmitochondria is negligible. This notion is supported by: (i)the inhibition of catalase does not predispose rat heart toinjury induced either by ischemia-reperfusion or H2O2 per-fusion [39], but (ii) overexpression of catalase by 60-fold[40], a value that according to our results renders catalasemore efficient than glutathione peroxidase in H2O2 re-moval, does increase resistance to ischemia-reperfusion;(iii) glutathione peroxidase knockout mice are more sus-ceptible to ischemia-reperfusion [41], while (iv) overex-pression of glutathione peroxidase increases resistance toischemia-reperfusion [42].

Fig. 3. Relative contributions of glutathione peroxidase and catalase to H2O2 removal in heart mitochondria. �GPx/(�GPx � �cat)(primary y-axis) and �cat/(�GPx � �cat) (secondary y-axis) were based on Eqn. 4 using the parameters obtained experimentally in thiswork: [GPxtot]/(�1kcat) � 44 and �2/�1 � 4.8 � 102. The effects of GSH depletion are shown for a range of H2O2 concentrations (10�9

to 10�3 M). Control levels for GSH (arrow) and a depletion of up to 90% are also indicated.


Physiological function of catalase

Catalase is an unusual enzyme with dual activities:besides eliminating H2O2 (reactions 4 and 5; catalatic ac-tivity), the enzyme can catalyze the oxidation of smallcompounds (reactions 4, 6, and 7; peroxidatic activity) [1].

catalase-Fe3� � H2O2 ¡k4

compound I (4)

compound I � H2O2 ¡k5

catalase-Fe3� � 2H2O � O2 (5)

compound I � AH2 ¡k6

catalase-Fe3� � 2H2O � A (6)

compound I � 2•NO � 2OH� O¡k7

catalase-Fe3�

� 2NO2� � H� (7)

It may be suggested that the physiological function ofthe low levels of catalase in heart mitochondria is relatedto its peroxidatic rather than its catalatic activity. Theclassic substrates for the peroxidatic activity are smallalcohols [1], but recently nitric oxide (•NO) was alsoidentified as a substrate [34] (Reaction 7). Because mi-cromolar concentrations of •NO strongly inhibited con-sumption of millimolar amounts of H2O2 by catalase[43], it may be assumed that the rate constant for theinteraction of •NO with compound I of catalase (k7) is1000-fold higher than that with H2O2 (k5). The catalaseactivity determined in this work (kcat) can be related withk5 by the following expression

kcat � 2k5[compound I]maximal (5)

The maximal fraction of catalase as compound I,which is obtained in the absence of peroxidatic substratesand under conditions of maximal turnover, is in the range0.3 to 0.5 [44]. Assuming k7 � 1000 k5, it may beobtained:

1000 kcat � 2k7�compound I]maximal (6)

The rate of •NO utilization by catalase in mitochon-dria is given by

� � NO � k7�compound I��•NO� (7)

Combining Eqns. 6 and 7, the rate of •NO utilizationby catalase can be estimated for different concentrationsof compound I, as shown in Table 2. In spite of the orderof magnitude calculation involved in these estimates, andbased on the comparison with the reaction with ubiquinoland superoxide anion [45,46], which are two of the mostimportant reactions of •NO in mitochondria [47], theoxidation of •NO with catalase is probably of physiolog-ical relevance.

CONCLUDING REMARKS

Altogether these observations imply that the detox-ification of H2O2 in heart mitochondria is a processthat occurs at expense of GSH oxidation via glutathi-one peroxidase, and that catalase does not have a rolein the elimination of H2O2. The eventual identificationof the physiological substrate of heart rat catalasemust involve an experimental quantitative analysisthat compares the oxidation catalyzed by catalase withthe oxidation of the same substance by competingenzymes. The calculations herein suggest that •NOmay be a likely physiological substrate for catalase inheart mitochondria.

Table 2. Comparison of the Rates of Utilization of •NO by Catalase with Competing Reactions in Heart Mitochondria

Reaction[compound I]/

[compound I]maximal k7 [compound I] Rate (�M s�1)c

1a 1.5 � 104 s�1

(1000 kcat)b

450

•NO � compound I 3 NO2� � catalase-Fe3�

0.1a 1.5 � 103 s�1

(100 kcat)b

45

0.01a 1.5 � 102 s�1

(10 kcat)b

4.5

•NO � UQH� 3 NO� � UQ•� – – 0.19d

•NO � O2•� 3 ONOO� – – 0.057d

a The maximal fraction of catalase in the form of compound I is assumed to be 0.5.b kcat determined in this work was 1.5 � 10�2 s�1 (mg of protein)�1 (in 1 ml reaction volume). Assuming 1 mg of protein/1 �l of mitoplast volume

we obtain a kcat of 15 s�1 after converting the units to mitoplast volume.c [•NO] is assumed to be 3 � 10�8 M (see [47]).d See [47] for the estimation of these rates.


Acknowledgements — Research supported by National Institutes ofHealth grant 1RO1-AG16718.

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ABBREVIATIONS

GPx—glutathione peroxidaseGPxox—glutathione peroxidase oxidized formGPxred—glutathione peroxidase reduced formGPxtot—total glutathione peroxidaseGSH—glutathioneGSSG—glutathione disulfide


relative contributions of heart mitochondria glutathione peroxidase and catalase to h2o2...

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