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Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase Rina Saha, Moumita Bose, Sumit Sen Santara, Jayasree Roy, Rajesh K. Yadav, Subrata Adak Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India abstract article info Article history: Received 9 April 2013 Received in revised form 21 June 2013 Accepted 24 June 2013 Available online 2 July 2013 Keywords: Leishmania Heme protein Peroxidase Peroxynitrite Steady-state catalysis Rapid kinetics and mutation The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base cat- alyst for heterolytic cleavage of an O\O bond in H 2 O 2 . However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to inves- tigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H 2 O 2 is decreased by approx- imately ve orders of magnitude relative to wild type, which is consistent with electron donor oxidation data where the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rate of intermediates in the mutants is not signicantly lower than that for the wild type, indicating that the His68 has no major role in homolytic cleavage of an O\O bond in peroxynitrite. EPR spectroscopic data suggest that the transient intermediates formed by the reaction of LmP with H 2 O 2 exhibits an intense and stable signal similar to CCP Compound ES whereas in case of the reaction with peroxynitrite, this signal disappears, indicating that the transient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higher decay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactive species in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higher peroxynitrite degrading activity. © 2013 Elsevier B.V. All rights reserved. 1. Introduction NO rapidly reacts with the superoxide anion (O 2 - ) to produce peroxynitrite (ONOO - ) that is a potent oxidizing and nitrating agent [1]. Numerous biological compounds present in physiological systems have been shown to be altered by ONOO - [2]. DNA, protein and mem- brane lipids are the sensitive biotargets for ONOO - mediated oxidative damage [35]. ONOO - has been shown to be a critical factor in several inammatory disorders, including atherosclerosis, rheumatoid arthritis, myocardial dysfunction and autoimmune diabetes [610]. Thus, it is es- sential to nd out the scavenger of ONOO - within the cell. CO 2 rapidly scavenges ONOO - in biological system but its reaction product (ONOOCO 2 - ) is a stronger nitrating agent than peroxynitrous acid [11,12]. However, several reports suggest that the selenocysteine- containing glutathione peroxidase and peroxiredoxin catalyzes the de- toxication of ONOO - [13,14]. Several groups of researchers have shown that ONOO - rapidly reacts with heme containing proteins including hemoglobin [15], myoglobin [16], peroxidases [17], pseudoperoxidase [18], nitric oxide synthase [19], catalase [17], cytochrome c [20], cytochrome P450 [21], and cyto- chrome c oxidase [22]. The synthetic ferric iron porphyrins have been shown to catalyze the isomerization of ONOO - to nitrate in vitro [23,24] and to be cytoprotective against ONOO - in vivo [25]. In addition, kinetic studies of the reaction of ONOO - with metmyoglobin and methe- moglobin suggest that the ferric iron forms of these proteins catalyze the isomerization of ONOO - to nitrate [15,16,26]. Kinetic evidence suggests that metmyoglobin reacts with ONOO - to form ferryl myoglobin and NO 2 [27] via initial formation of a caged radical intermediate [Fe IV = ONO 2 ] and this caged pair reacts mainly via internal return to form metMb and NO 3 - in an oxygen rebound scenario. Unlike the synthetic ferric iron porphyrins, metmyoglobin and methemoglobin, ONOO - re- acts with various peroxidases and converts native enzymes to the quasi-stable and catalytically inactive form of Compound II [17]. Thus, it is quite clear that the nature of the intermediate has been an important point of debate in the synthetic ferric iron porphyrins and heme proteins work, which remains to be solved unambiguously. Heme peroxidases rapidly react with H 2 O 2 to produce Compound I or Compound ES that contains Fe IV = O and a cation radical [2831]. The initial step is believed to be the formation of peroxidaseperoxo complexes [32] that undergo very rapid heterolytic O\O bond cleavage with the help of a widely conserved distal histidine [3335]. Similarly, the reaction of the Fe III state of heme peroxidase with ONOO - produces transient intermediates [17] but nothing is known regarding the role of the active site residues of peroxidase in ONOO - dependent transient Biochimica et Biophysica Acta 1834 (2013) 20572063 Abbreviations: LmP, Leishmania major peroxidase; HRP, horseradish peroxidase; CCP, cytochrome c peroxidase; OONO - , peroxynitrite; Compound I, Fe(IV) = O heme with a porphyrin radical; Compound ES, Fe(IV) = O heme with an amino acid radical Corresponding author. Tel.: +91 33 2473 6793; fax: +91 33 2473 5197. E-mail address: [email protected] (S. Adak). 1570-9639/$ see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.06.018 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap

Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase

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Biochimica et Biophysica Acta 1834 (2013) 2057–2063

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbapap

Effect of distal His mutation on the peroxynitrite reactivity ofLeishmania major peroxidase

Rina Saha, Moumita Bose, Sumit Sen Santara, Jayasree Roy, Rajesh K. Yadav, Subrata Adak ⁎Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700 032, India

Abbreviations: LmP, Leishmania major peroxidase;CCP, cytochrome c peroxidase; OONO−, peroxynitrite; Cwith a porphyrin radical; Compound ES, Fe(IV) = O hem⁎ Corresponding author. Tel.: +91 33 2473 6793; fax

E-mail address: [email protected] (S. Adak).

1570-9639/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbapap.2013.06.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 April 2013Received in revised form 21 June 2013Accepted 24 June 2013Available online 2 July 2013

Keywords:LeishmaniaHeme proteinPeroxidasePeroxynitriteSteady-state catalysisRapid kinetics and mutation

The conserved distal histidine in peroxidases has been considered to play amajor role as a general acid-base cat-alyst for heterolytic cleavage of an O\O bond in H2O2. However, heme peroxidases react with peroxynitrite toform transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to inves-tigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E,H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H2O2 is decreased by approx-imately five orders of magnitude relative to wild type, which is consistent with electron donor oxidation datawhere the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rateof intermediates in the mutants is not significantly lower than that for the wild type, indicating that the His68has no major role in homolytic cleavage of an O\O bond in peroxynitrite. EPR spectroscopic data suggest thatthe transient intermediates formed by the reaction of LmPwithH2O2 exhibits an intense and stable signal similarto CCP Compound ESwhereas in case of the reactionwith peroxynitrite, this signal disappears, indicating that thetransient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higherdecay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactivespecies in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higherperoxynitrite degrading activity.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

NO rapidly reacts with the superoxide anion (O2−) to produce

peroxynitrite (ONOO−) that is a potent oxidizing and nitrating agent[1]. Numerous biological compounds present in physiological systemshave been shown to be altered by ONOO− [2]. DNA, protein and mem-brane lipids are the sensitive biotargets for ONOO−mediated oxidativedamage [3–5]. ONOO− has been shown to be a critical factor in severalinflammatory disorders, including atherosclerosis, rheumatoid arthritis,myocardial dysfunction and autoimmune diabetes [6–10]. Thus, it is es-sential to find out the scavenger of ONOO− within the cell. CO2 rapidlyscavenges ONOO− in biological system but its reaction product(ONOOCO2

−) is a stronger nitrating agent than peroxynitrous acid[11,12]. However, several reports suggest that the selenocysteine-containing glutathione peroxidase and peroxiredoxin catalyzes the de-toxification of ONOO− [13,14].

Several groups of researchers have shown that ONOO− rapidly reactswith heme containing proteins including hemoglobin [15], myoglobin[16], peroxidases [17], pseudoperoxidase [18], nitric oxide synthase

HRP, horseradish peroxidase;ompound I, Fe(IV) = O hemee with an amino acid radical

: +91 33 2473 5197.

rights reserved.

[19], catalase [17], cytochrome c [20], cytochrome P450 [21], and cyto-chrome c oxidase [22]. The synthetic ferric iron porphyrins have beenshown to catalyze the isomerization of ONOO− to nitrate in vitro[23,24] and to be cytoprotective against ONOO− in vivo [25]. In addition,kinetic studies of the reaction of ONOO−withmetmyoglobin andmethe-moglobin suggest that the ferric iron forms of these proteins catalyze theisomerization of ONOO− to nitrate [15,16,26]. Kinetic evidence suggeststhat metmyoglobin reacts with ONOO− to form ferryl myoglobin andNO2 [27] via initial formation of a caged radical intermediate [FeIV =O•NO2] and this caged pair reacts mainly via internal return to formmetMb and NO3

− in an oxygen rebound scenario. Unlike the syntheticferric iron porphyrins, metmyoglobin and methemoglobin, ONOO− re-acts with various peroxidases and converts native enzymes to thequasi-stable and catalytically inactive form of Compound II [17]. Thus,it is quite clear that the nature of the intermediate has been an importantpoint of debate in the synthetic ferric iron porphyrins and heme proteinswork, which remains to be solved unambiguously.

Heme peroxidases rapidly react with H2O2 to produce Compound Ior Compound ES that contains FeIV = O and a cation radical [28–31].The initial step is believed to be the formation of peroxidase–peroxocomplexes [32] that undergo very rapid heterolytic O\O bond cleavagewith the help of a widely conserved distal histidine [33–35]. Similarly,the reaction of the FeIII state of heme peroxidase with ONOO− producestransient intermediates [17] but nothing is known regarding the role ofthe active site residues of peroxidase in ONOO− dependent transient

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intermediates formation. However, pseudoperoxidase from Leishmaniamajor (LmPP) lacking the distal histidine displays lower peroxidase ac-tivity and higher ONOO− decomposition activity than the V90Hmutantof LmPP [18].

In the present work, mutagenesis is used to simplify the mecha-nism of heterolytic and homolytic O\O bond cleavage in H2O2 andONOO−, respectively, by creating a L. major peroxidase mutant thatlacks the distal His residue. The mutations of distal His-68 to Valand Glu display 3–5 orders of magnitude decreased rate of the hetero-lytic O\O bond cleavage in H2O2 but do not significantly change therate of the homolytic O\O bond cleavage in ONOO−. Our present ex-periments have indicated that the rate of ONOO− decomposition isincreased by distal His mutation. A detailed characterization of theONOO− decomposition reaction of the wild type LmP, His68Val andHis68Glu mutant is reported here.

2. Materials and methods

2.1. Materials

ONOO− was obtained from Calbiochem, La Jolla, CA, as an aque-ous solution (180–200 mM) and stored frozen at −80 °C until use.The stock solution was diluted with 0.005 M NaOH, and the con-centration of ONOO− was measured spectrophotometrically be-fore each experiment by measuring the absorbance at 302 nm(ε302 = 1705 M−1 cm−1). Horse cytochrome c, potassium iodide,guaiacol and imidazole were obtained from Sigma-Aldrich. The sourcesof other reagents were described previously [18,36–38].

2.2. Molecular biology

Site-directed mutagenesis of Δ34LmP DNA in the pTrcHisA expres-sion plasmid (coding for amino acids 34–303 plus a six-His tag at theN terminus) was performed using the QuikChange site-directed muta-genesis kit from Stratagene. Themutation codon (bold)was incorporat-ed into the primers as follows: sense H68V, 5′-CGCCTCGCATGGGTGGAGGCCGCCTCG-3′; antisense H68V, 5′-CGAGGCGGCCTCCACCCATGCGAGGCG-3′; and sense H68E, 5′-CGCCTCGCATGGGAGGAGGCCGCCTCG-3′;antisense H68E, 5′-CGAGGCGGCCTCCTCCCATGCGAGGCG-3′. Themuta-tionswere confirmed at themolecular biology core facility of the IndianInstitute of Chemical Biology.

2.3. Expression and purification of wild-type and mutant LmP

Wild-type and mutant enzymes were overexpressed in Escherichiacoli BL21 D3 and purified by using Ni2+-nitrilotriacetate affinity chro-matography as reported earlier [39]. Concentrations of wild type,H68V and H68E enzymes were determined from the 406 nm absor-bance of the heme, using an extinction coefficient of 101, 104 and110 mM−1 cm−1, respectively.

2.4. Peroxidase activity measurement

H2O2-driven ascorbate, guaiacol and cytochrome c oxidation by LmP(wild type and mutant) were assayed as described earlier [39]. In short,0.1 μM enzymes were incubated with 0.5 mM ascorbate or 20 mMguaiacol at 50 mM phosphate buffer pH 7.5 in a final volume of 1.0 ml.In case of cytochrome c oxidation, 10 nM enzyme was incubated with50 μM ferrous horse cytochrome c at 50 mM phosphate buffer pH 7.5.Reactions were initiated by adding 0.3 mM H2O2. The rate of ascorbate,guaiacol or cytochrome c oxidation was determined from the change ofabsorbance at 290 nm, 470 nm, or 550 nm respectively. The concentra-tions of oxidized ascorbate, guaiacol and cytochrome c were determinedfrom ε290 = 2.8 × 103 M−1 cm−1, ε470 = 2.6 × 104 M−1 cm−1 andε550 = 2.1 × 104 M−1 cm−1, respectively.

2.5. Rapid scanning experiments by stopped-flow spectrophotometry

The time-resolved spectra were collected from 340 to 700 nm onHi-Tech stopped-flow instrument (KinetAsyst), using a rapid scanningdiode array device (Hi-TechMG-6560) designed to record 300 completespectra within 450 ms in each mixing. Rapid scanning experiments in-volved mixing solutions containing high spin enzyme in 50 mM phos-phate buffer, pH 7.5 with varying concentrations of ONOO− or H2O2

solutions at 25 °C. Kinetics of the formation of the transient intermedi-ates wasmeasured at 420 nmand signal-to-noise ratios were improvedby averaging 10 individual traces. Average traces were fit to singleexponential functions using software provided by the instrument man-ufacturer. Second order rate constants for the transient intermediateformation of LmP were obtained by the plotting of kobs versus varyingconcentrations of ONOO− or H2O2.

The decay rates of ONOO− in the presence of wild type andmutantswere studiedwith the stopped-flow instrument. Themixing time of theinstrument is about 0.75 ms. The kinetic traces were collected at340 nm instead of 302 nm for the decay rate of ONOO− [18]. Themolar extinction coefficient of ONOO− is 868 at 340 nm and 1705 at302 nm. For the detection of transient intermediates, the protein solu-tions in the presence of 100 mM phosphate buffer pH 6.0 or 7.5 weremixed with varying concentrations of ONOO−. The kinetic traces werecollected at 421 nm for the formation rate of transient intermediates.The results of the fits of the traces (averages of at least 10 single traces)from at least three experiments were averaged to obtain each observedrate constant, given with the corresponding standard deviation. ONOO−

solutions were prepared by diluting the stock solution instantly beforeuse with 0.005 M NaOH to get the required concentration.

2.6. Electron paramagnetic resonance measurements

X-band EPR spectra weremeasured on a Jeol (JESFA 200) apparatus.LmP (0.4 mM)was dissolved in 50 mMphosphate buffer (pH 7.5)withH2O2 (0.4 mM) or ONOO− (0.8 mM) for the measurement of EPR. EPRsamples were prepared by combining 0.4 mM enzyme with 0.4 mMH2O2 or 0.8 mMONOO−. Those samples were immediately transferredto an EPR tube and flash frozen in liquid nitrogen within 3 min.

3. Results

3.1. Mutant LmP purification and spectroscopic characterization

LmPmutantswere purified in absence of electron donor and typical-ly yielded about 10 mg per liter of culture that was comparable to ouryield of wild-type LmP expressed in the same system. The H68V andH68E LmP used in our experiment were purified to homogeneity andhad the same molecular mass as wild-type LmP (data not shown). TheUV–visible spectra of both His68 mutants showed the presence of aSoret peak at 406 nm with visible peaks at ~500 and 640 nm. Spectro-scopic data revealed that both His68 mutants contained heme in a pre-dominantly high spin state, which closely resembles that of wild type(Fig. 1A–C). The H2O2 mediated spectral changes were studied byrapid scan UV/vis spectroscopy between 340 and 700 nm at pH 7.0and 25 °C to obtain a spectrum of the intermediate over the entirewavelength range. As shown in Fig. 1A–B, the Soret bands of wild typeand H68E mutant LmP shifted from 406 nm to 420 nm. The final spec-trum inH68Emutantwas reminiscent of Compound ES of thewild typeLmP due to α and β band in visible region at 560 and 532 nm [36].These results are consistent with previous CCP and HRP results[40,41]. In contrast with the wild type LmP, H2O2 is unable to shift theSoret band from 406 nm to 420 nm in the H68Vmutant upon additionof H2O2 at 50 mM phosphate buffer pH 7.0 (Fig. 1C). Although H68Eand H68Vmutants react with 20 equivalent of H2O2 with increased ab-sorbance at 421 nm, the change in absorbance at 421 nmduring the re-action with H2O2 is 88% for H68E and 9% for H68V compared to wild

Fig. 1. Electronic absorption spectra and kinetics of Compound ES formation inwild type andmutants LmP. Panels A, B, and C represent the spectra of H2O2 driven transient intermediatesinwild type, H68E andH68Vmutant, respectively. Those spectrawere obtained by the rapidmixing between4.0 μMof enzyme and4.0 μMH2O2 forwild type or 100 μMH2O2 formutantsat 25 °C in 50 mM phosphate buffer pH 7.0. The dotted line, the solid line and the dashed line represent before, after short time and, after long time interval of addition of H2O2, respec-tively. Panels D, E and F represent the rate of Compound ES formation in wild type H68E and H68V mutant, respectively. Reactions are initiated by rapid mixing of each of the 4 μM ofenzyme in 50 mM phosphate buffer, pH 7.0 with an equal volume of buffer solution containing an equimolar concentration of H2O2. Smooth lines drawn through the kinetic tracesare the calculated lines of best fit according to one exponential reaction. Data in each panel are representative of an average of 10 individual reactions. Insets show the rate of CompoundES decay in wild type, H68E and H68V mutant.

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type LmP (Fig. 1A–C). These data showed that the distal site mutationaffected the heterolytic cleavage of H2O2 to form the transient interme-diate Compound ES in LmP. On the other hand, addition of two timesmolar excess ONOO− to the resting state of wild type and mutantsLmP produced the transient intermediates absorbing at 420 nm at theSoret regionwith visible peaks at 532 and 560 nm(Fig. 2A–C). These re-sults suggest that the distal site His residue does not have anymajor rolein homolytic cleavage of the O\O bond of the ONOO− for the genera-tion of ferryl intermediates.

3.2. EPR spectroscopy

To identify the nature of ONOO−driven transient intermediates,we utilized electron paramagnetic resonance spectroscopy to followheme transitions during H2O2 or ONOO−-driven transient intermedi-ates formation in wild type LmP (Fig. 3). Electron donor free-ferricLmP was mixed with equimolar H2O2 or two fold molar excess ofONOO− at 25 °C in presence of 50 mM phosphate buffer pH 7.5. Inthe presence of H2O2, LmP exhibits a very intense signal centered atg = 1.9975 (Fig. 3) with the peak-to-trough line width = 24.2 G,consistent with Trp radical formation [31]. In contrast, ONOO−-drivenEPR signal in LmP is basically lost (Fig. 3). Moreover, its UV visible op-tical spectrum of ONOO−-driven reaction with LmP (Fig. 3 inset)clearly differed from the pea ascorbate peroxidase Compound I (ferrylporphyrin π cation radical) that formed as an immediate product byH2O2 [29]. Thus, during ONOO−-driven transient intermediates for-mation of electron donor free LmP, a ferryl species was formed with-out formation of stable porphyrin or protein radicals. Therefore,ONOO−-driven transient intermediates in LmP might be CompoundII rather than Compound ES.

3.3. Kinetic studies for the formation and decay of the intermediate inH2O2 treated wild type and mutant protein

Kinetic studies for the formation of transient Compound ES fromelectron donor free high spin protein were carried out by followingthe absorbance changes at 421 nm at pH 7.0. As shown in Fig. 1D–F,the measured traces showed a rapid increase in absorption. The for-mation was fitted to a single-exponential expression to give kobs. Asexpected, the rate of formation, measured within a broad range ofH2O2 concentrations, was linearly concentration dependent. Asshown in Table 1, the second order rate constants for CompoundES formation in H68E and H68V were more than 3–5 orders of mag-nitude less than that in wild type. Compound ES was much shorterlived in each of the H68E, and H68V mutants than in wild type(Fig. 1D–F inset). Like CCP distal site His mutants [40], the non-stoichiometric formation of Compound ES for our two LmP mutantscould be due to the slow formation of Compound ES followed byrapid decay. In the reaction between H2O2 and H68E LmP shownin Fig. 1B, the Soret band shifted from 406 nm to 420 nm duringthe initial part of the reaction, characteristic of Compound ES forma-tion, followed by a return to 408 nm as Compound ES decays. Theendogenous decay of Compound ES for wild type and His mutantsLmP was monitored by decreasing absorbance at 421 nm. The kinet-ic traces were described by a monophasic rate constant, and the en-dogenous decay of Compound ES for wild type, H68E and H68Vmutants LmP were 0.0002 ± 0.00001, 0.085 ± 0.01 and 0.018 ±0.002 s−1, respectively. Thus, distal His68 appears to have a dualrole in peroxidase catalysis; one, facilitating Compound ES forma-tion with the reaction of H2O2 by acting as an acid-base catalystand the other is the stabilization of Compound ES by inhibitingendogenous decay of Compound ES.

Fig. 2. Electronic absorption spectra and kinetics of ONOO− driven Compound II formation in wild type and mutants LmP. Panels A, B, and C represent the spectra of ONOO− driventransient intermediates in wild type, H68E and H68V mutant, respectively. Those spectra were obtained by the rapid mixing between 8.0 μMONOO− and 4.0 μM of enzyme at 25 °Cin 50 mM phosphate buffer pH 6.0. The dotted line, the solid line and the dashed line represent before, after short time and after long time interval of addition of ONOO−, respec-tively. Panels D, E and F represent the rate of Compound II formation in wild type, H68E and H68V mutant respectively. Reactions are initiated by rapid mixing of each of the 4 μM ofenzyme in 50 mM phosphate buffer, pH 6.0 with an equal volume of buffer solution containing two fold molar excess ONOO−. Smooth lines drawn through the kinetic traces arethe calculated lines of best fit according to one exponential reaction. Data in each panel are representative of an average of 10 individual reactions. Insets show the rate of Com-pound II decay in wild type, H68E and H68V mutant.

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3.4. Spectroscopy and kinetics of wild type and His68 mutant reactionwith ONOO−

To better understand the characteristics of transient intermediates,we utilized rapid-scanning stopped-flow spectroscopy to follow heme

Fig. 3. Electron paramagnetic resonance spectral analysis of the reaction of LmPwithH2O2

or ONOO−. X-band EPR spectra of 0.4 mM native LmP protein (solid line), 0.4 mM H2O2

treated (dashed line) or 0.8 mM ONOO−-treated (dotted line) protein in 50 mM phos-phate buffer, pH 7.5, containing 0.1 mM DTPA. The microwave power, modulation fre-quency, microwave frequency, and measurement temperature were 5.0 mW, 100 kHz,9.1 GHz, and 77 K, respectively. The small signal around g = 2 is due to cavity contamina-tion in wild type and ONOO− treated LmP. Inset shows optical spectra of same samplesafter EPR experiment.

transitions during ONOO−-driven intermediate formation in LmP(Fig. 2A–C). The EPR results suggested that transient intermediates inthe ONOO− driven reaction in wild type were Compound II (withoutradical formation). Fig. 2A–C shows the change in the resting state ofhigh spin wild type or mutant spectrum when a two fold molar excessof ONOO− was added in presence of 100 mM phosphate bufferpH-6.0. The spectrum of wild type and mutants were red-shifted to420 nm at the Soret region with α and β bands at 560 and 527 nm.The spectrum of the intermediate of wild type enzyme at pH 6.0 wasobserved within ~50 ms upon mixing with ONOO− and was almostidentical to ferryl form observed in H2O2-dependent Compound II inHRP [32]. After Compound II formation, the Soret band reverted backfrom 420 nm to the starting FeIII spectrum 406 nmwithin few secondsin mutant proteins. These results suggest that the endogenous reduc-tion of ferryl iron in distal histidine mutants might be faster than wildtype LmP. In the crystal structures of Compound I for HRP and Com-pound ES for CCP, the water molecule forms a H-bonding bridge be-tween the ferryl O atom and the distal His. Thus it is very likely thatthese sets of interactions help to stabilize the ferryl center [42,43].

We next analyzed the kinetics of the Compound II formation in thewild type andmutants LmP. Fig. 2D–F depicted the kinetics of CompoundII formation during the reaction between enzyme and ONOO− at 25 °Cfor wild type and mutants LmP. The reactions were started by rapidmixing between a solution of ONOO− and LmP. The absorbance increaseat 421 nm for all proteins was best fitted to a one-exponential equation,giving apparent second order rate constants listed in Table 1, suggestingthat the apparent second order rate constant of the reactivity betweenenzyme and ONOO− was not significantly altered by the mutation ofHis68 to Val or Glu. The increased rates of decay at 421 nm in the mu-tants indicate that the distal His is important in stabilizing Compound II(Fig. 2D–F inset). The kinetic traces were described by monophasic rate

Table 1Observed rate constants for the formation and decay of transient intermediates in presence of H2O2 or ONOO− at 25 °C. Reactions are initiated by rapidmixing the enzymewith differentconcentrations of H2O2 or ONOO− as described under “Materials andmethods.” The formation rates of transient intermediates describe the buildup of absorbance at 421 nm versus timeand are the average obtainedwith two enzyme preparations. The data best fit to one-exponential curve in all cases to generate rate constant (kobs). The second order rate constants for theformation of transient intermediates are calculated from linear curve of kobs versus H2O2 or ONOO− concentration. The decay (reduction) rates of transient intermediates describe thedecrease in absorbance at 421 nmversus time and are the average obtainedwith two enzyme preparations. The data bestfit to one-exponential curve in all cases to generate rate constant(kobs).

LmP Compound ES by H2O2 at pH 7.0 Compound II by ONOO− at pH 6.0

Formation Decay Formation Decay

Rate (M−1 s−1) % × 10−2 (s−1) Rate (M−1 s−1) % × 10−2 (s−1)

Wild type 9.6 ± 0.4 × 107 100 0.02 ± 0.001 4.5 ± 1.0 × 106 100 0.19 ± 0.01H68V 1.4 ± 0.02 × 102 9 8.5 ± 1 1.5 ± 0.7 × 106 97 10 ± 0.6H68E 8.9 ± 0.7 × 104 88 1.8 ± 0.2 2.6 ± 0.2 × 106 96 14 ± 1.0

Fig. 4. Reaction of ONOO−withwild type, H68E and H68V LmP. Panels upper,middle andlower represent the decay rate of ONOO− (400 μM) bywild type, H68E and H68Vmutantprotein in the presence of 100 mM phosphate buffer pH 6.0, respectively. Thick linesdrawn through the kinetic traces are the calculated lines of best fit according to one expo-nential reaction. Insets show plots of kobs vs protein concentration at different pH.

2061R. Saha et al. / Biochimica et Biophysica Acta 1834 (2013) 2057–2063

constant, and the endogenous decay of the ferryl form of heme iron formutants LmP was ~50–70 fold faster than wild type (Table 1).

3.5. Kinetic studies for the ONOO− decomposition at different pH by wildtype and mutant proteins

The rate of ONOO− decomposition in the presence or absence of thedifferent type of LmP proteinswasmeasured by stopped-flow spectros-copy at pH 6.0 or pH 7.5. The rate of reactionswas calculated by follow-ing the decrease in absorbance changes at 340 nm instead of 302 nmdue to wavelength limitation of our stopped-flow spectrophotometer.The absorbance change could be fitted well to a one-exponential equa-tion. As shown in Fig. 4A–C, the observed ONOO− decomposition ratesin wild type LmP (kobs) were lower than in mutant proteins. For com-parison, we determined the rates of ONOO− decomposition in the pres-ence of various amounts of the protein (Fig. 4A–C inset). The kobs valuesincreased linearly with increasing protein concentrations. On the basisof the turnover number of ONOO− degradation, kcat, expressed as s−1

in the absence of H2O2 (Table 2), wild type was found to be less effi-cient catalyst than mutants. As expected, at a lower pH-6.0, thevalues for kcat were greater than higher pH 7.5 (Fig. 4A–C inset,Table 2), and mutants were still found to be the most efficient cata-lysts compared to wild type.

3.6. Multiple turnover rate of electron donor oxidation

We compared peroxidase activity among wild type and mutants bymeasuring their multiple turnover rates of H2O2-dependent ascorbate,guaiacol and ferrous horse cytochrome c oxidation. Like earlier CCPand HRP results [33,34,44], the ascorbate, guaiacol and cytochrome coxidations were ~100–1000 fold lower in the mutants compared withwild-type in the presence of 0.3 mM H2O2 (Table 2). Thus the catalyticactivities were in line with the observation of kinetic results of Com-pound ES formation where the rate of Compound ES formation inwild type was found to be faster compared to distal H68 mutants. TheTable 2 depicted that the distal histidine in LmP played a crucial rolein differentiation between peroxidase and ONOO− decompositionactivities.

4. Discussion

In general, it is established that the main role of the distal histidinein most of the peroxidases is to serve as a base catalyst, facilitatingheterolytic cleavage of the oxygen-oxygen bond of H2O2, resultingin the formation of active intermediate Compound ES. Then it cancarry out a variety of reduced electron donor oxidation [33,34,45].In addition, it plays a crucial role in stabilizing the ferryl heme ironas well as in endogenous reduction back to the FeIII state [40]. TheX-ray structure of LmP shows that the His 68 residue is present inthe distal site of heme [31]. It is not surprising that mutation of LmP

Table 2Catalytic activities of wild type, H68E and H68V LmP at 25.0 °C. Enzymes were purified from overexpression system. Wild type and mutants enzymes were purified three timesindependently and catalytic activities were determined as described under “Materials and methods.”.

LmP enzyme Decay of peroxynitritea Ascorbateb

kcat (s−1)Guaiacolb

kcat (s−1)Cytochrome cb

kcat (s−1)pH-6.0kcat (s−1)

pH-7.5kcat (s−1)

Wild type 0.8 ± 0.01 0.05 ± 0.002 1.4 ± 0.2 6.0 ± 0.5. 28 ± 1H68E 13 ± 1.1 2.6 ± 0.1 1.2 ± 0.1 × 10−1 3.3 ± 0.1 × 10−1 6.9 ± 2 × 10−1

H68V 21 ± 1.8 5.1 ± 0.3 1.6 ± 0.1 × 10−2 3.1 ± 0.15 × 10−3 3.2 ± 0.1 × 10−2

a The turnover number of ONOO− degradation is expressed as s−1 in the absence of H2O2. Reactions were initiated by rapidly mixing 4.0 mM enzyme in the presence of 100 mMphosphate buffer pH 7.5 or pH 6.0, with 400 mM peroxynitrite in 0.005 N NaOH. The molar extinction coefficient of ONOO− is 868 at 340 nm.

b The turnover number of electron donor oxidation is expressed as s−1 in presence of 0.3 mM H2O2. The concentrations of ascorbate, guaiacol and ferrous horse cytochrome cused were 500 μM, 20 mM and 50 μM, respectively. The values represent the mean and standard deviation for three measurements each.

Fig. 5. Proposed reaction scheme that can describe the observed data of wild-type andmutant LmP. Negative sign indicates inhibition.

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His 68 (identical position of CCP His-52) to Glu or Val will influence therates of the Compound ES formation and stabilization. However, therole of the distal histidine in the ONOO− dependent Compound ESand II formation of peroxidase is still obscured. If the distal histidineserves as a base catalyst for both H2O2 and ONOOH cleavage, then therates of these two reactions should change in a similar manner for allof the LmP mutants that alter the basicity of the residue at position 68in LmP. At neutral pH, there is no reasonably good correlation betweenH2O2 reaction and ONOO− reaction for the LmP mutants. Although thesecond order rate constant for the ONOO− dependent Compound II for-mation in distal His mutants is similar to wild type LmP protein; theH2O2 dependent Compound ES formation rate in distal histidine mu-tants takes place ~3–5 orders of magnitude slower (Table 2) than inwild type LmP. On the other hand, the ferryl form of iron state (gener-ated by H2O2 or ONOO−) in wild type LmP is more stable than distalHis mutants in the absence of added reducing agent. This result is con-sistent with the previous distal site His mutant results in CCP and HRP[40,41]. Several key points in this manuscript are: (a) homolytic cleav-age of the O\O bond of ONOO− by LmP results in the formation of tran-sient intermediate Compound II rather than Compound ES, (b) thesecond order rate constant for ONOO− dependent Compound II forma-tion in mutant proteins are similar compared to wild type enzyme, (c)the rate of endogenous reduction back from Fe_O to the Fe (III) stateinmutants protein is ~103 times higher compared towild type enzyme,and (d) the substitution of His residue in LmP protein shows hyperac-tive enzyme with respect to ONOO− decomposition.

The next question is why the initial reaction rate with ONOO− is notinfluenced by the distal His residue in LmP? The reactivity of H2O2 withheme proteins is different compared to the reactivity of ONOO− withheme protein. However, it has been demonstrated that the distal histi-dine in heme protein catalyzes deprotonation of the H2O2 assisting thebinding of the peroxide anion to the heme iron [40] but the pH depen-dence of the catalytic rate constants shows that protonation of theONOO− helps the binding of the HOONO to the heme iron of the pro-teins [46]. Like our distal histidine mutant proteins, previously severalgroup of workers have reported by using cytochrome P450s and syn-thetic water soluble metalloporphyrins, which lack distal histidine,that ONOO− reacts rapidly with heme iron to form Fe_O species[23,47,48].

X-ray crystal structures of Compound I in HRP show that the watermolecule derived from the distal oxygen atom is within hydrogen bond-ing distance of the oxygen atom remaining on the iron [43]. This wateralso forms hydrogen bonds with the Nε2 atom of His 42, the side-chainof Arg 38 and anotherwatermolecule [43]. Similarly the crystal structureof CCP Compound ES shows a water molecule forming a H-bondingbridge between the ferryl O atom and the distal His [42]. Like HRP andCCP, the glutamate substitution at distal His 68 residue in LmP also desta-bilizes Fe_O species compared to the wild-type enzyme and theoxyferryl-FeIV site reduced ~280 times faster in the mutant. The reduc-tion rate of Fe_O species, which is generated by ONOO−, is similar tothe reduction rate of Fe_O species that is generated by H2O2 in H68ELmP mutant.

A central focus of this study iswhy the catalysis of ONOO− decompo-sition is more in the distal histidine mutants compared to the wild typeLmP. The work presented in this manuscript demonstrates convincinglythat distal His residue in LmP differentiates the peroxidative catalysisinstead of the ONOO− isomerization activity. On the basis of previousresults in met myoglobin with ONOO− [16], the transient intermediate[FeIV = O•NO2] can either proceed to cage escape or rebound to formNO3

− and ferric Mb [48]. In LmP, the enzyme rapidly reacts withONOO− and the enzyme may quickly partition between a catalyticallyactive ferric form and an inactive ferryl form (Compound II). Thiscreates two cycles having their own rate-limiting steps and together de-termine the observed rate of ONOO− decomposition during the steady-state (Fig. 5). A given LmP molecule circulates in the active or inactivecycles depending on whether ferryl heme rebinds •NO2 to form NO3

and active ferric LmP or cage escapes •NO2 to form inactive ferryl LmP.The unambiguous observation of the ferrylMb intermediate providesstrong confirmation of the proposed mechanism, in which twomechanisms may be formulated to interpret our results (Fig. 5). Therate-limiting step in the active cycle, which leads to ONOOH break-down, could be ONOOH binding, a homolytic O\O cleavage stepinvolved in FeIV = O•NO2 formation, or •NO2 dissociation, or •NO2

rebinding to form NO3− and ferric enzyme. It is still unclear which of

these is the rate-limiting for wild-type LmP, but our work clearlyshows that the rate of Compound II formation (FeIV = O) is unalteredwith distal histidine mutation. We postulate that the oxyferryl state ofheme iron is the inactive state of LmP during enzyme catalyzed ONOO−

decomposition and the rate-limiting step becomes the stability of Com-pound II. Our results suggest that the stability of Compound II in wildtype LmP is 70 times higher than mutant enzymes. Now, it is clear whysynthetic metalloporphyrin and pseudoperoxidase (lacks distal histi-dine) would be favored in the ONOO− decomposition [18,46,48] com-pared to electron donor free peroxidase [17,49].

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Acknowledgements

This work was supported by Council of Scientific and IndustrialResearch (CSIR) Project BSC 0114, ICMR fellowship to R.S, CSIR fel-lowships to M.B. and R.K.Y., CSIR-SPM fellowships to S. S. S.

References

[1] T. Nauser, W.H. Koppenol, The rate constant of the reaction of superoxide with ni-trogen monoxide: approaching the diffusion limit, J. Phys. Chem. A 106 (2002)4084–4086.

[2] P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health anddisease, Physiol. Rev. 87 (2007) 315–424.

[3] H. Ischiropoulos, J.S. Beckman, Oxidative stress and nitration in neurodegeneration:cause, effect, or association? J. Clin. Invest. 111 (2003) 163–169.

[4] T. Douki, J. Cadet, Peroxynitrite mediated oxidation of purine bases of nucleosidesand isolated DNA, Free Radic. Res. 24 (1996) 369–380.

[5] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite-induced membranelipid peroxidation: the cytotoxic potential of superoxide and nitric oxide, Arch.Biochem. Biophys. 288 (1991) 481–487.

[6] H. Kaur, B. Halliwell, Evidence for nitric oxide-mediated oxidative damage inchronic inflammation nitrotyrosine in serum and synovial fluid from rheumatoidpatients, FEBS Lett. 350 (1994) 9–12.

[7] C.R. White, T.A. Brock, L.Y. Chang, J. Crapo, P. Briscoe, D. Ku, W.A. Bradley, S.H.Gianturco, J. Gore, B.A. Freeman, et al., Superoxide and peroxynitrite in athero-sclerosis, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 1044–1048.

[8] N.W. Kooy, S.J. Lewis, J.A. Royall, Y.Z. Ye, D.R. Kelly, J.S. Beckman, Extensive tyro-sine nitration in human myocardial inflammation: evidence for the presence ofperoxynitrite, Crit. Care Med. 25 (1997) 812–819.

[9] W.L. Suarez-Pinzon, C. Szabo, A. Rabinovitch, Development of autoimmune diabe-tes in NOD mice is associated with the formation of peroxynitrite in pancreaticislet beta-cells, Diabetes 46 (1997) 907–911.

[10] C. Szabo, H. Ischiropoulos, R. Radi, Peroxynitrite: biochemistry, pathophysiologyand development of therapeutics, Nat. Rev. Drug Discov. 6 (2007) 662–680.

[11] S.V. Lymar, J.K. Hurst, Role of compartmentation in promoting toxicity ofleukocyte-generated strong oxidants, Chem. Res. Toxicol. 8 (1995) 833–840.

[12] W.A. Pryor, J.N. Lemercier, H. Zhang, R.M. Uppu, G.L. Squadrito, The catalytic roleof carbon dioxide in the decomposition of peroxynitrite, Free Radic. Biol. Med. 23(1997) 331–338.

[13] H. Sies, V.S. Sharov, L.O. Klotz, K. Briviba, Glutathione peroxidase protects againstperoxynitrite-mediated oxidations. A new function for selenoproteins asperoxynitrite reductase, J. Biol. Chem. 272 (1997) 27812–27817.

[14] R. Bryk, P. Griffin, C. Nathan, Peroxynitrite reductase activity of bacterial peroxiredoxins,Nature 407 (2000) 211–215.

[15] S. Herold, M. Exner, T. Nauser, Kinetic and mechanistic studies of the NO*-mediatedoxidation of oxymyoglobin and oxyhemoglobin, Biochemistry 40 (2001) 3385–3395.

[16] S. Herold, S. Kalinga, T. Matsui, Y. Watanabe, Mechanistic studies of the isomeri-zation of peroxynitrite to nitrate catalyzed by distal histidine metmyoglobin mu-tants, J. Am. Chem. Soc. 126 (2004) 6945–6955.

[17] R. Floris, S.R. Piersma, G. Yang, P. Jones, R. Wever, Interaction of myeloperoxidasewith peroxynitrite A comparison with lactoperoxidase, horseradish peroxidaseand catalase, Eur. J. Biochem. 215 (1993) 767–775.

[18] M. Bose, R. Saha, S. Sen Santara, S. Mukherjee, J. Roy, S. Adak, Protection againstperoxynitrite by pseudoperoxidase from Leishmania major, Free Radic. Biol.Med. 53 (2012) 1819–1828.

[19] J.P. Pasquet, M.H. Zou, V. Ullrich, Peroxynitrite inhibition of nitric oxide synthases,Biochimie 78 (1996) 785–791.

[20] A.M. Cassina, R. Hodara, J.M. Souza, L. Thomson, L. Castro, H. Ischiropoulos, B.A.Freeman, R. Radi, Cytochrome c nitration by peroxynitrite, J. Biol. Chem. 275(2000) 21409–21415.

[21] Y. Minamiyama, S. Takemura, S. Imaoka, Y. Funae, Y. Tanimoto, M. Inoue, Irrevers-ible inhibition of cytochrome P450 by nitric oxide, J. Pharmacol. Exp. Ther. 283(1997) 1479–1485.

[22] M.A. Sharpe, C.E. Cooper, Interaction of peroxynitrite with mitochondrial cyto-chrome oxidase catalytic production of nitric oxide and irreversible inhibitionof enzyme activity, J. Biol. Chem. 273 (1998) 30961–30972.

[23] M.K. Stern, M.P. Jensen, K. Kramer, Peroxynitrite decomposition catalysts, J. Am.Chem. Soc. 118 (1996) 8735–8736.

[24] J.B. Lee, J.A. Hunt, J.T. Groves, Mechanisms of iron porphyrin reactions withperoxynitrite, J. Am. Chem. Soc. 120 (1998) 7493–7501.

[25] T.P. Misko, M.K. Highkin, A.W. Veenhuizen, P.T. Manning, M.K. Stern, M.G. Currie,D. Salvemini, Characterization of the cytoprotective action of peroxynitrite de-composition catalysts, J. Biol. Chem. 273 (1998) 15646–15653.

[26] N. Romero, R. Radi, E. Linares, O. Augusto, C.D. Detweiler, R.P. Mason, A. Denicola,Reaction of human hemoglobin with peroxynitrite isomerization to nitrate andsecondary formation of protein radicals, J. Biol. Chem. 278 (2003) 44049–44057.

[27] J. Su, J.T. Groves, Direct detection of the oxygen rebound intermediates, ferryl Mband NO2, in the reaction of metmyoglobin with peroxynitrite, J. Am. Chem. Soc.131 (2009) 12979–12988.

[28] J.E. Penner-Hahn, K.S. Eble, T.J. McMury, M. Renner, A.L. Balch, J.T. Groves, J.H.Dawson, K.O. Hodgson, Structural characterization of horseadish peroxidaseusing EXAFS spectroscopy, evidence for Fe_O ligation in compounds I and II, J.Am. Chem. Soc. 108 (1986) 7819–7825.

[29] W.R. Patterson, T.L. Poulos, D.B. Goodin, Identification of a porphyrin pi cationradical in ascorbate peroxidase compound I, Biochemistry 34 (1995) 4342–4345.

[30] M. Sivaraja, D.B. Goodin, M. Smith, B.M. Hoffman, Identification by ENDOR ofTrp191 as the free-radical site in cytochrome c peroxidase compound ES, Science245 (1989) 738–740.

[31] V.S. Jasion, J.A. Polanco, Y.T. Meharenna, H. Li, T.L. Poulos, Crystal structure ofLeishmania major peroxidase and characterization of the compound I tryptophanradical, J. Biol. Chem. 286 (2011) 24608–24615.

[32] H.B. Dunford, Heme Peroxidases, John Wiley & Sons, Inc., New York, 1999.[33] J.E. Erman, L.B. Vitello, M.A. Miller, A. Shaw, K.A. Brown, J. Kraut, Histidine 52 is a

critical residue for rapid formation of cytochrome c peroxidase compound I, Bio-chemistry 32 (1993) 9798–9806.

[34] S.L. Newmyer, P.R. Ortiz de Montellano, Horseradish peroxidase His-42 – N Ala,His-42 – N Val, and Phe-41 – N Ala mutants histidine catalysis and control of sub-strate access to the heme iron, J. Biol. Chem. 270 (1995) 19430–19438.

[35] J.N. Rodriguez-Lopez, A.T. Smith, R.N.F. Thornley, Recombinant horseradish per-oxidase isoenzyme C: the effect of distal haem cavity mutations (His42 → Leuand Arg38 → Leu) on compound I formation and substrate binding, J. Biol.Inorg. Chem. 1 (1996) 136–142.

[36] R.K. Yadav, S. Dolai, S. Pal, S. Adak, Role of tryptophan-208 residue in cytochrome coxidation by ascorbate peroxidase from Leishmania major-kinetic studies onTrp208Phe mutant and wild type enzyme, Biochim. Biophys. Acta 1784 (2008)863–871.

[37] R.K. Yadav, S. Pal, S. Dolai, S. Adak, Role of proximal methionine residues in Leish-mania major peroxidase, Arch. Biochem. Biophys. 515 (2011) 21–27.

[38] S. Mukherjee, S. Sen Santara, S. Das, M. Bose, J. Roy, S. Adak, NAD(P)H cytochromeb5 oxidoreductase deficiency in Leishmania major results in impaired linoleatesynthesis followed by increased oxidative stress and cell death, J. Biol. Chem.287 (2012) 34992–35003.

[39] S. Adak, A.K. Datta, Leishmania major encodes an unusual peroxidase that is aclose homologue of plant ascorbate peroxidase: a novel role of the transmem-brane domain, Biochem. J. 390 (2005) 465–474.

[40] M.C. Foshay, L.B. Vitello, J.E. Erman, Effect of alternative distal residues on the re-activity of cytochrome c peroxidase: properties of CcP mutants H52D, H52E,H52N, and H52Q, Biochim. Biophys. Acta 1814 (2011) 525–535.

[41] M. Tanaka, K. Ishimori, M. Mukai, T. Kitagawa, I. Morishima, Catalytic activitiesand structural properties of horseradish peroxidase distal His42 – N Glu or Glnmutant, Biochemistry 36 (1997) 9889–9898.

[42] C.A. Bonagura, B. Bhaskar, H. Shimizu, H. Li, M. Sundaramoorthy, D.E. McRee, D.B.Goodin, T.L. Poulos, High-resolution crystal structures and spectroscopy of nativeand compound I cytochrome c peroxidase, Biochemistry 42 (2003) 5600–5608.

[43] G.I. Berglund, G.H. Carlsson, A.T. Smith, H. Szoke, A. Henriksen, J. Hajdu, The cat-alytic pathway of horseradish peroxidase at high resolution, Nature 417 (2002)463–468.

[44] L. Lad, M. Mewies, J. Basran, N.S. Scrutton, E.L. Raven, Role of histidine 42 in ascor-bate peroxidase. Kinetic analysis of the H42A and H42E variants, Eur. J. Biochem.269 (2002) 3182–3192.

[45] T.L. Poulos, J. Kraut, The stereochemistry of peroxidase catalysis, J. Biol. Chem. 255(1980) 8199–8205.

[46] R. Saha, M. Bose, S. Adak, Mutation of Val90 to His in the pseudoperoxidase fromLeishmania major enhances peroxidase activity, Biochim. Biophys. Acta 1834(2013) 651–657.

[47] R. Shimanovich, J.T. Groves, Mechanisms of peroxynitrite decomposition cata-lyzed by FeTMPS, a bioactive sulfonated iron porphyrin, Arch. Biochem. Biophys.387 (2001) 307–317.

[48] J. Su, J.T. Groves, Mechanisms of peroxynitrite interactions with heme proteins,Inorg. Chem. 49 (2010) 6317–6329.

[49] S.C. Grace,M.G. Salgo,W.A. Pryor, Scavenging of peroxynitrite by a phenolic/peroxidasesystem prevents oxidative damage to DNA, FEBS Lett. 426 (1998) 24–28.