Original Contribution

MODULATION OF CATALASE PEROXIDATIC AND CATALATIC ACTIVITYBY NITRIC OXIDE

LUCA BRUNELLI,*†‡ VLADIMIR YERMILOV,§ and JOSEPHS. BECKMAN§

*Division of Neonatal Medicine, Duke University Medical Center, Durham, NC, USA;†Division of Anesthesia and IntensiveCare, Giannina Gaslini Children’s Hospital, Genoa, Italy;‡Department of Pediatrics, University of Turin, Turin, Italy; and

§Department of Anesthesiology, The University of Alabama at Birmingham, Birmingham, AL, USA

(Received7 December2000;Accepted12 December2000)

Abstract—Previously, we found that catalase enhanced the protection afforded by superoxide dismutase toEscherichiacoli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al.,Arch. Biochem. Biophys.316:327–334, 1995). Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxidedismutase. We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide.Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosylcomplex. Catalase consumed more nitric oxide upon the addition of hydrogen peroxide. Conversely, micromolarconcentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide. Catalase pretreatedwith nitric oxide and hydrogen peroxide regained full activity after dialysis. Our results suggest that catalase can slowlyconsume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide. Theprotective effects of catalase in combination with superoxide dismutase may result from two actions; reducingperoxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts withsuperoxide dismutase to form additional superoxide. © 2001 Elsevier Science Inc.

Keywords—Nitric oxide, Catalase, Antioxidants, Hydrogen Peroxide, Free radicals

INTRODUCTION

Catalase is a four subunits 240 kDa ferric hemoproteinthat mediates the two step decomposition of hydrogenperoxide (H2O2) to water [1]. This is commonly referredto as thecatalaticactivity of catalase and occurs accord-ing to the following pathway:

catalase-Fe31 1 H2O2 1 2H1

f compound I~Fe41 5 O! 1 H2O (1)

compound I~Fe41 5 O! 1 H2O2

f catalase-Fe31 1 H2O 1 O2 (2)

Catalase also demonstrates aperoxidaticactivity to-ward small substrates, such as methanol, ethanol, for-mate, and azide as first reported by Keilin and Hartree[2,3]. The essential feature of the peroxidatic activity ofcatalase is the oxidation of these alternative substrates incompetition with hydrogen peroxide for compound I(reaction 2). Therefore, the peroxidatic activity is mostevident at relatively low concentrations of hydrogen per-oxide [3,4]. Catalase prefers small substrates because theheme groups are deeply buried and are accessible onlyby a narrow channel lined with hydrophobic residues [5].

Nitric oxide (•NO) is a small hydrophobic gas that hasphysiologic functions as diverse as smooth muscle relax-ation, platelet inhibition, neurotransmission, and stimu-lation of hormone release [6–8]. The information carriedby nitric oxide is translated into a cellular signal byreversibly binding to the ferrous heme iron of guanylatecyclase that stimulates cGMP synthesis [7]. However,nitric oxide can also react with ferric hemoproteins suchas ferrimyoglobin and ferric horseradish peroxidase [9].

Present address and address correspondence to: Luca Brunelli, M.D.,Children’s Hospital at Strong, University of Rochester Medical Center,601 Elmwood Avenue, PO Box 777, Rochester, NY 14642, USA; Tel:(716) 275-0747; Fax: (716) 442-6580; E-Mail: luca_brunelli@urmc.rochester.edu.

Free Radical Biology & Medicine, Vol. 30, No. 7, pp. 709–714, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/01/$–see front matter

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The restrictive active site of catalase is large enoughto allow ready access to nitric oxide. Catalase is knownto form a transient complex with nitric oxide when itoxidizes azide in the presence of hydrogen peroxide [10].We became interested in the interactions of nitric oxidewith catalase when we observed that catalase signifi-cantly decreased the bactericidal activity of 3-morpholi-nosydnonimine-N-ethylcarbamide (SIN-1), a compoundthat produces peroxynitrite (ONOO2) through the re-lease of nitric oxide and superoxide (O2

•2) [11]. There-fore, we investigated the effects of nitric oxide on thecatalase-mediated scavenging of hydrogen peroxide andwhether nitric oxide might be consumed by the peroxi-datic activity of catalase.

MATERIALS AND METHODS

A 1.9 mM nitric oxide solution was prepared bybubbling 100 ml of water in a 300 ml gas samplingcylinder (Fisher Scientific, Fair Lawn, NJ, USA) withargon for 15 min and then with nitric oxide for 5 min.Catalase was purchased from Worthington BiochemicalCorp., Freehold, NJ, USA. Hydrogen peroxide (30%)was obtained from Fisher Scientific. Catalase was dia-lyzed against 50 mM potassium phosphate, pH 7.0, andfilter sterilized. Spectral analysis was carried out witheither a UV-260 Shimadzu or a 8452A Hewlett Packarddiode array spectrophotometer. Nitric oxide was mea-sured by a nitric oxide chemiluminescent detector devel-oped in our laboratory as previously described [12]. Inbrief, nitric oxide was measured in a well-stirred solution

by allowing it to diffuse into a microporous polypro-pylene membrane that was connected to a Model 705Antek Instruments (Houston, TX, USA) chemilumines-cent detector. Nitric oxide was added to a solution in thedetecting chamber followed by catalase after 15 s, andthen hydrogen peroxide. All experiments were carriedout in 50 mM potassium phosphate, pH 7.0. The resultsare the mean of four to five experiments.

RESULTS

The Soret band of native catalase peak shifted from406 to 426 nm with an isosbestic point at 415 nm when20 mM nitric oxide was added to the catalase solutionunder either aerobic or anaerobic conditions (Fig. 1).Two new peaks appeared at 540 nm and at 575 nm. Suchspectral changes are in agreement with those reported byNicholls for the nitric oxide-catalase complex and aretypical of a ferrous-nitrosyl complex [10]. Nitric oxidewas then removed from the catalase solution by bubblingargon, which slowly regenerated the spectrum of nativecatalase and restored full enzymatic activity.

Catalase alone produced a stoichiometric decrease innitric oxide concentration, consistent with the binding offour nitric oxides per catalase tetramer (Fig. 2). Althoughnitric oxide readily forms a complex with the restingferric state of catalase, nitric oxide could also inhibitcatalase under high turnover conditions in the presenceof a 10,000-fold excess of hydrogen peroxide. Micromo-lar concentrations of nitric oxide strongly inhibited theconsumption of millimolar hydrogen peroxide by cata-

Fig. 1. Reversible formation of nitric oxide-catalase complex. The spectrum of native catalase is shown in curve A. Curve B wasobtained after the addition of 20mM nitric oxide to catalase. Identical results were obtained when the experiment was carried out underanaerobic conditions. To remove nitric oxide, the sample was transferred to a beaker with argon passed over the surface for 10 min.The resulting spectrum obtained is shown in curve C. The solution was then again bubbled with argon for 10 min. The results are shownin curve D.

710 L. BRUNELLI et al.

lase (Fig. 3). Inhibition followed simple hyperbolic ki-netics with respect to nitric oxide concentration and wasmore pronounced with lower concentrations of hydrogenperoxide. The inhibition of 20mM nitric oxide on cata-lase activity was maximal in the first minute and thendecreased spontaneously (Fig. 4), most likely due to the

removal of nitric oxide by its reaction with dissolvedoxygen. The addition of 100 mM ethanol, an alternativesubstrate for catalase, did not affect inhibition by nitricoxide (data not shown).

DISCUSSION

Catalase readily forms a complex with nitric oxide,and Brown [13] showed that submicromolar concentra-tions of nitric oxide inhibited oxygen evolution by cata-lase. In the present study, we showed that nitric oxide inaddition substantially slowed the overall consumption ofhydrogen peroxide. Oxygen evolution only assesses thesecond step in the catalase cycle (reaction 2) and iscompetitively inhibited by alternative peroxidative sub-strates like ethanol. However, many peroxidative sub-strates like ethanol actually stimulate overall hydrogenperoxide consumption by catalase by increasing the turn-over of compound I and thereby minimizing the forma-tion of inhibitory complexes such as compound III [14].Unlike ethanol, nitric oxide competitively inhibited hy-drogen peroxide decomposition. Nitric oxide can readilyform a complex with the resting ferric state of catalasethat may account in part for the observed inhibition.However, equivalent inhibition was observed when nitricoxide was added to catalase either before or after addinglarge excesses of hydrogen peroxide, suggesting thatnitric oxide might further be capable of forming aninhibitory complex with compound I.

Brown [13] also showed that catalase could consumenitric oxide in the presence of hydrogen peroxide, whichwe confirmed. The oxidation product of nitric oxide ismost likely nitrite:

Fig. 2. The peroxidatic activity of catalase on nitric oxide. The signal was generated by the addition of 10mM nitric oxide to thedetecting chamber. Subsequently, 0.2mM catalase and 5 mM hydrogen peroxide were added. There was no reaction between hydrogenperoxide and nitric oxide in the absence of catalase.

Fig. 3. Inhibition of catalase-mediated consumption of hydrogen per-oxide by nitric oxide. The influence of increasing concentrations ofnitric oxide on the rate of catalase-mediated decomposition of hydro-gen peroxide was assayed by the decrease in absorbance at 240 nm of10 mM hydrogen peroxide over 30 s. The mean rates of hydrogenperoxide consumption for at least four different experiments were thenplotted vs. the nitric oxide concentration. The concentration of catalasewas held constant at 0.34 nM to allow for a linear consumption ofhydrogen peroxide over the first 30 s. Nitric oxide was present in themedium with hydrogen peroxide and the reaction was started by addingcatalase. The order of addition made no difference in the inhibition.

711Nitric oxide modulates catalase

Compound I~Fe41 5 O! 1 •NO 1 OH2

f catalase-Fe31 1 NO22 1 H1 (3)

The ability of catalase to rapidly scavenge nitric oxidecomplicates the interpretation of in vitro experimentswhere inhibition by catalase has traditionally been takenas strong evidence for the toxicity of hydrogen peroxide.The lifetime of biologically relevant concentrations ofnitric oxide can be as long as hours in simple buffers andeven in hemoglobin-free tissue homogenates [15]. Invivo, hemoglobin in red blood cells as well as myoglobinprovides effective sinks that scavenge nitric oxide, lim-iting its half-life to less than a second. Here we haveshown that nitric oxide is capable of outcompeting hy-drogen peroxide for catalase. Therefore, catalase couldrepresent a potential sink for nitric oxide, particularlywhen added to cell culture experiments as a hydrogenperoxide scavenger that otherwise lacks a means to con-sume nitric oxide.

We became interested in this possibility when wefound that catalase substantially decreases the toxicity ofSIN-1 towardsE. coli even in the presence of 100 to1000 U/ml SOD [11]. In theory, SOD should have scav-enged the superoxide release by SIN-1 and thereby in-creased hydrogen peroxide formation. However, wewere unable to measure significant accumulation (. 1mM) of hydrogen peroxide, which was far too low forhydrogen peroxide to be directly responsible for bacterialkilling in this system [11].

Previously, we have shown that nitric oxide is stillbeing consumed at a rapid rate by SIN-1 even in the

presence of high concentrations of SOD [15]. This couldnot simply be explained by the faster reaction of super-oxide with nitric oxide compared to SOD. BecauseSIN-1 decomposition continuously produces nitric oxide,the concentration of nitric oxide rises after a fixed con-centration of SOD is added to SIN-1 until the greaterconcentration of nitric oxide allows it to again competewith SOD for superoxide. Our results and those ofBrown [13] suggest that catalase can potentially decreaseperoxynitrite formation by providing an alternative routeto scavenge nitric oxide. Catalase limits the buildup ofnitric oxide by utilizing some of the hydrogen peroxideformed by SOD-catalyzed dismutation to oxidize nitricoxide. Minimizing the buildup of NO thereby allowsSOD to compete more efficiently with nitric oxide forsuperoxide (Fig. 5).

Gergel et al. [16] showed that hydrogen peroxideconcentrations were kept at low levels during SIN-1decomposition in the presence of SOD. In their studies ofSIN-1 toxicity to a hepatic cell line, they suggested thatthe formation of hydroxyl radical by the following slowreaction of hydrogen peroxide with reduced copper inSOD accounted for the toxicity.

Cu11SOD1 H2O2 N Cu21SOD1 HO 2 1 HO•

(4)

However, hydroxyl radical generated outside thebacterial wall is not toxic toE. coli [17], and in ourhands hydroxyl radical scavengers did not affect thetoxicity of SIN-1 plus SOD toE. coli. More recently,

Fig. 4. Time-course of the inhibition of the catalatic activity of catalase under different orders of addition of nitric oxide. The effectof nitric oxide on the ability of catalase to decompose 10 mM hydrogen peroxide was studied over 4 min. Catalase concentration waskept constant at 2.9 nM, nitric oxide concentration at 20mM, and hydrogen peroxide concentration at 10 mM. The dotted line wasobtained after addition of nitric oxide to hydrogen peroxide. Catalase was added last to start the reaction mixture. In the continuousline tracing, the reaction between catalase and hydrogen peroxide was started, then after about 30 s nitric oxide was added. The shiftin baseline level in such tracing was due to the absorbance of nitric oxide and its decomposition products at 240 nm.

712 L. BRUNELLI et al.

the generation of bicarbonate radical has been shownto be an important intermediate in the SOD-peroxidereaction and may be a significant toxin to mammaliancells [18,19].

However, there is an additional means involving hy-drogen peroxide by which catalase can indirectly reducethe formation of peroxynitrite (Fig. 5). When SOD isadded to SIN-1, the steady state levels of hydrogenperoxide remain in the submicromolar concentrationrange because it reacts at a modest but significant ratewith SOD [16].

H2O2 N H1 1 HO22 (5)

HO22 1 Cu21SODN O2

•2 1 Cu11SOD1 H1 (6)

Cu11SOD1 O2 N Cu21SOD1 O2•2 (7)

The reaction of HO22 is substantially faster for Cu21

versus Cu11SOD, making the regeneration of superoxidefrom hydrogen peroxide substantially more favorablethan the formation of hydroxyl radical. In the absence ofcatalase, this hydrogen peroxide is gradually convertedback to superoxide by the large amount of oxidized SOD.In the presence of nitric oxide, hydrogen peroxide plusSOD generates peroxynitrite as elegantly shown byMcBride et al. [20]. At physiologically realistic concen-trations of hydrogen peroxide (micromolar and below)the reduced copper left by hydrogen peroxide will slowlyautooxidize to produce additional superoxide that in turncan produce additional peroxynitrite [21,22]. Althoughnitric oxide inhibits catalase, substantial catalase activityremains at the levels of nitric oxide produced in theSIN-1 experiments that minimize these secondary reac-tions of hydrogen peroxide with SOD. Consequently, theprotective effects observed with catalase against SIN-1-induced toxicity may depend upon the combined actions

of scavenging both hydrogen peroxide and nitric oxide tominimize the formation of peroxynitrite. This furthersupports the importance of interlocking antioxidant de-fenses where SOD has been shown to protect catalasefrom inactivation by superoxide and catalase to protectCu,Zn SOD from hydrogen peroxide [14].

Acknowledgements—We thank Dr. Irwin Fridovich for providing lab-oratory space, equipment and many helpful discussions. Dr. Brunellithanks Prof. Alberto Bertolini and Prof. Alberto Ponzone for theirsupport.

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Fig. 5. Scheme to show how catalase can reduce the formation of peroxynitrite from SIN-1 by scavenging nitric oxide. In addition,catalase removes hydrogen peroxide, preventing it from reacting with oxidized SOD to regenerate superoxide. Reduced SOD also issusceptible to autooxidation and can produce an additional superoxide. Consequently, catalase can have two distinct actions to reducethe formation of peroxynitrite.

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