Free Radical Biology & Medicine, Vol. 37, No. 6, pp. 813 –822, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2004.06.006

Original Contribution

INACTIVATION OF HUMAN Cu,Zn SUPEROXIDE DISMUTASE BY

PEROXYNITRITE AND FORMATION OF HISTIDINYL RADICAL

BEATRIZ ALVAREZ,*,y VERONICA DEMICHELI,*,y,z ROSARIO Duran,§ MADIA TRUJILLO,y,z

CARLOS CERVENANSKY,§ BRUCE A. FREEMAN,b and RAFAEL RADIy,z

*Laboratorio de Enzimologıa, Facultad de Ciencias, Universidad de la Republica, 11400 Montevideo, Uruguay; zDepartamento deBioquımica, Facultad de Medicina, Universidad de la Republica, 11800 Montevideo, Uruguay; yCenter for Free Radical andBiomedical Research, Uruguay; § Instituto de Investigaciones Biologicas Clemente Estable 11600 Montevideo, Uruguay; and

bDepartments of Anesthesiology, Biochemistry, and Molecular Genetics, and Center for Free Radical Biology,University of Alabama at Birmingham, Birmingham, AL 35294, USA

(Received 13 January 2004; Revised 26 May 2004; Accepted 4 June 2004)

Available online 25 June 2004

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Abstract—Human recombinant copper–zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the

product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the

activity by 50% (IC50) was f100 AM at 5 AM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow

determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was

(9.4 F 1.0) � 103 M�1 s�1 per monomer at pH 7.5 and 37jC. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5

mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of

an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal

consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol

on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen

peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that

peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts

with histidine residues, forming histidinyl radical. D 2004 Elsevier Inc. All rights reserved.

Keywords—Peroxynitrite, Superoxide dismutase, Histidinyl radical, Superoxide, Nitric oxide, Free radicals

INTRODUCTION

The enzyme copper–zinc superoxide dismutase (CuZn-

SOD) catalyzes the disproportionation of superoxide

anion to dioxygen and hydrogen peroxide [1]. This

antioxidant enzyme is present in the cytosol and mito-

chondrial intermembrane space of eukaryotic cells and

in the periplasmic space of bacterial cells as a homo-

dimer of 32 kDa. Each monomer binds one copper and

one zinc ion. The reaction mechanism involves the

ress correspondence to: Beatriz Alvarez, Laboratorio de Enzimo-

acultad de Ciencias, Igua 4225, 11400 Montevideo, Uruguay;

982 5250749; E-mail: Beatriz.Alvarez@fcien.edu.uy; or Rafael

epartamento de Bioquımica, Facultad de Medicina, Avda. Gral.

2125, 11800 Montevideo, Uruguay; Fax: +5982 9249563;

: rradi@fmed.edu.uy.

813

sequential reduction and reoxidation of Cu(II), for which

both reactions are relatively pH-independent and pro-

ceed with rate constants of 2 � 109 M�1 s�1:

OS�2 þ CuðIIÞZnSOD ! O2 þ CuðIÞZnSOD;

OS�2 þ CuðIÞZnSODþ 2Hþ ! H2O2 þ CuðIIÞZnSOD:

The Cu(II) ion is bound to four histidine residues. One of

them also coordinates the Zn(II) ion, together with two

more histidines and one aspartate.

Several point mutations of CuZnSOD have been

implicated in the devastating motor neuron disease

familial amyotrophic lateral sclerosis (ALS) [2]. It is

generally accepted that the disease is initiated by the

gain of a new and toxic property [3]. Candidates for

B. ALVAREZ et al.814

this deleterious property are an increased ability to

catalyze oxidations [4] and to induce protein aggrega-

tion [5–7].

Hydrogen peroxide, or rather its conjugate base

(HO2�), reacts with CuZnSOD, reducing Cu(II) to

Cu(I), followed by the reaction of Cu(I) with a second

hydrogen peroxide forming an active site oxidant, puta-

tively described as a copper-bound hydroxyl radical [8].

This in turn leads to enzyme inactivation through 2-

oxohistidine formation [9] and to the oxidation of various

substrates in what is called the ‘‘peroxidative’’ activity of

CuZnSOD [10–13]. Carbon dioxide enhances this activ-

ity through the intermediacy of the longer lived carbonate

radical [14–16]. Recently, the histidinyl radical was

detected as the intermediate in the reaction of CuZnSOD

with hydrogen peroxide through electron paramagnetic

resonance spectroscopy (EPR) with 2-methyl-2-nitroso-

propane (MNP) as spin trap [17].

Increasing evidence points to a role in ALS for

peroxynitrite,1 the product of the diffusion-controlled

reaction between nitric oxide and superoxide. In this

sense, elevated levels of nitrotyrosine, a relatively spe-

cific marker of peroxynitrite formation, were detected in

both sporadic and familial forms of ALS [18,19]. It has

been proposed that in ALS, CuZnSOD could lose zinc

and form peroxynitrite through the reaction of nitric

oxide with superoxide produced from the reduced en-

zyme reacting with dioxygen [20].

Peroxynitrite is mostly (80%) anionic at pH 7.4. Its

conjugate acid (pKA 6.8) can homolyze at a rate of 0.9

s�1 at pH 7.4 and 37jC, forming nitrogen dioxide and

hydroxyl radicals in f30% yield. The preferential bio-

targets of peroxynitrite are metal centers, sulfur and

selenium compounds, and carbon dioxide, although tar-

gets that do not react directly with peroxynitrite, such as

tyrosine, may nevertheless be modified by the radicals

formed from it (reviewed in [21]). The reactions of

peroxynitrite with transition metal centers are some of

the fastest known for peroxynitrite. It has been proposed

that, in the same way as with other Lewis acids such as a

proton or carbon dioxide, the reaction proceeds to form

an adduct that in turn homolyzes to yield nitrogen

dioxide and the corresponding oxyradical [21,22]:

ONOO� þMnþ ! ONOO��Mnþ !SNO2 þ�SO�Mnþ

!SNO2 þ OjMðnþ1Þþ:

In the case of CuZnSOD, this general mechanism

allows us to rationalize the fact that the enzyme is able to

increase the yield of nitrotyrosine formation from perox-

1 The term peroxynitrite is used to refer to both peroxynitrite anion

and peroxynitrous acid. IUPAC-recommended names are oxoperoxoni-

trate (1-) and hydrogen oxoperoxonitrate, respectively.

ynitrite, most likely through the intermediacy of free

radical species [23–25]. Accordingly, mitochondrial

MnSOD increases the yield of nitration of self and

remote tyrosines, with the site-specific nitration of tyro-

sine 34 leading to enzyme inactivation [26–28].

In this paper, we investigate the reactivity of peroxyni-

trite toward CuZnSOD; we study the kinetics, the inacti-

vation of the enzyme, and the formation of protein free

radicals, and we discuss our observations in light of the

biochemistry known for bothCuZnSODand peroxynitrite.

MATERIALS AND METHODS

Materials

Peroxynitrite solutions were prepared from acidified

hydrogen peroxide and sodium nitrite in a tandem

quenched-flow mixing apparatus [29]. The solutions

were treated with manganese dioxide to remove residual

hydrogen peroxide. The concentration of peroxynitrite

was determined from its absorbance at 302 nm (q = 1.67

mM�1 cm�1 [30]). Stock solutions were stored at�80jC, used only once after thawing, and then dis-

carded. The concentration of nitrite present as contam-

inant was measured with the Griess reagent [31,32] after

decay of peroxynitrite to nitrate in monobasic sodium

phosphate solution. Nitrite content was measured in

every experiment and was always less than 30% of

peroxynitrite. Hydrogen peroxide was from J.T. Baker

and its concentration was determined from the absor-

bance at 240 nm (q = 43.6 M�1 cm�1 [33]). Xanthine

oxidase was from Calbiochem. Bovine CuZnSOD was

from Grunenthal. All other reagents were from Sigma.

Purification of human recombinant wild-type CuZnSOD

A pET-3d plasmid encoding wild-type human re-

combinant CuZnSOD (kind gift from Dr. J.S. Beckman,

Oregon State University) [25] was expressed in Escher-

ichia coli strain BL21(DE3)plysS. The bacterial cells

were transformed by the calcium chloride method [34].

Cultures were grown in Luria broth containing ampi-

cillin and chloramphenicol at 37jC. CuZnSOD expres-

sion was induced by adding 1 mM isopropyl-h-D-thiogalactopyranoside. ZnCl2 (0.2 mM) and CuCl2(0.2 mM) were also added. After 4 h at 24jC, cells

were harvested by centrifugation, resuspended in 20

mM Tris–HCl, pH 7.8, and frozen overnight. The

suspension was then thawed, sonicated, treated with

DNase, and centrifuged for 15 min at 18,000 � g.

CuZnSOD was purified through ammonium sulfate

fractionation followed by ion-exchange chromatography

in Sepharose CL6B. After elution with a gradient of 0–

200 mM NaCl in Tris 2 mM, pH 7.4, a single

polypeptide chain was visible in SDS–PAGE, and a

single peak with the expected molecular mass of

Peroxynitrite inactivation of CuZnSOD 815

15,796 F 10 was seen through MALDI-TOF mass

spectrometry. To improve the specific activity, the pro-

tein was incubated overnight at 4jC with zinc and

copper salts [25], followed by gel filtration on Sephadex

G-75. Further remetallation with zinc ion added before

copper ion at pH 5.5 [35] did not increase the specific

activity of the preparation. CuZnSOD was concentrated

by ultrafiltration, washed with 10 vol of sodium phos-

phate, 20 mM, pH 7.4, and stored frozen.

Protein and metal determinations

The protein concentration was determined through the

enhanced BCA assay (Pierce) using bovine CuZnSOD as

standard. Similar results were obtained using bovine

serum albumin as standard. The protein concentration

determined with the BCA assay differed less than 30%

from absorbance at 280 nm (q = 5400 M�1 cm�1 for the

monomer [36,37]) or Biuret determinations. The protein

had a specific activity of (2.1 F 0.4) � 103 U mg�1. The

zinc and copper content was determined through the 4-

pyridylazoresorcinol assay in the presence of guanidine

[25] using atomic absorption standard solutions for

calibration and found to be 0.94 F 0.05 and 1.00 F0.07 ions per monomer for zinc and copper, respectively.

In the absence of guanidine, the concentration of non-

specifically bound metals was found to be less than 0.04

and 0.06 ions per monomer.

Superoxide dismutase activity

The dismutase activity2 was measured through the

decrease in the rate of the superoxide-dependent cyto-

chrome c reduction at 550 nm using xanthine/xanthine

oxidase as superoxide source [1,38]. Activity was also

assessed in native 15% polyacrylamide gels stained

with 4-nitroblue tetrazolium (NBT) [38]. The bands

were revealed through the reduction of NBT (0.25 mg/

ml) with superoxide produced by the photochemical

reduction of riboflavin (0.1 mg/ml) with TEMED

(1%).

2 The activity was determined by plotting 1/v, the reciprocal of the

rate, vs. the microliters of CuZnSOD added to the 1 ml assay cuvette,

fitting to a linear plot through the least-squares method and determining

the units per milliliter by dividing the slope by the y-axis intercept:

U mL�1¼ 1000

3

slope

y � interceptdilution factor

Unless otherwise specified, the relative error in the activity

determination was estimated as the sum of the relative standard errors

(symbolized as D) in the slope and the y intercept as determined in the

least-squares fit:

DðU mL�1Þ ¼ U mL�1 D slope

slopeþ D y � intercept

y � intercept

� �

Exposure of CuZnSOD to peroxynitrite

Peroxynitrite was added to the enzyme dissolved in

0.05 M sodium phosphate buffer containing DTPA (0.1

mM), at pH 7.4 F 0.1, unless otherwise specified. After

3 min incubation at 37jC, the enzyme was put on ice and

diluted 20- or 30-fold for activity determination. Experi-

ments were repeated independently at least three times.

Phosphate buffers were prepared daily avoiding the use

of sodium hydroxide in order to minimize bicarbonate/

carbon dioxide contamination. For experiments in which

bicarbonate/carbon dioxide was specifically added, the

buffers were prepared immediately before the experiment

and used within 10 min to minimize diffusion of carbon

dioxide out of the solution [39].

Kinetics

The rate of the reaction between peroxynitrite and

CuZnSOD was determined in a stopped-flow spectro-

photometer (Applied Photophysics, SF17MV). The ap-

parent rate constant of peroxynitrite decay at 37jC was

determined at 302 nm in the presence of increasing

concentrations of CuZnSOD using an initial rate ap-

proach described previously [40]. The first 0.1–0.2 s

were fitted to a linear plot and the apparent rate constant

was determined as � (dA/dt)/(A0� Al), the ratio between

the slope and the difference between the initial and the

final absorbance. Two hundred absorbance measure-

ments were acquired during the first 0.2 s and 200 further

points were acquired until more than 99.9% of the

peroxynitrite had decomposed (0.2–10 s). The pH was

measured after the mixing of peroxynitrite with the

buffer, and two independent experiments were performed

with similar results.

Simulations

Chemical reaction simulations using the known rate

constants and initial conditions were performed with the

software Gepasi [41,42].

EPR experiments

The EPR spectra were recorded at room tempera-

ture with a Bruker EMX spectrometer. Spin trapping

experiments were performed by adding peroxynitrite (1

mM) to CuZnSOD (0.5 mM monomer) in reaction

mixtures containing phosphate buffer (0.1 M, pH 7.4),

DTPA (0.1 mM), and MNP (22 mM). The reaction

mixtures were transferred to 200 Al flat cells immedi-

ately after peroxynitrite addition and the acquisition of

the spectra was started within 1 min. Proteolysis was

performed by gel filtration of peroxynitrite-treated

CuZnSOD with MicroBio-Spin 6 chromatography col-

umns (Bio-Rad) followed by the addition of Pronase

(40 mg ml�1) and acetonitrile (14%). For comparison,

B. ALVAREZ et al.816

spectra of reaction mixtures containing free L-histidine

(100 mM) and peroxynitrite (7.1 mM) were acquired.

For some experiments, stock solutions of MNP (0.5

M) were prepared in acetonitrile (HPLC quality).

Computer simulations of EPR spectra were calculated

using the EPR-WinSim public software tool of NIEHS

(http://www.epr.niehs.nih.gov/).

RESULTS

Enzyme inactivation by peroxynitrite

Human recombinant wild-type CuZnSOD was inac-

tivated by peroxynitrite. As observed in Fig. 1, expo-

sure of CuZnSOD to increasing concentrations of

peroxynitrite led to a dose-dependent loss of the

dismutase activity. At an enzyme concentration of

Fig. 1. Superoxide dismutase activity of CuZnSOD exposed toperoxynitrite. (A) CuZnSOD (4.9 AM monomer) was exposed toincreasing concentrations of peroxynitrite in sodium phosphate buffer,0.05 M, pH 7.4, DTPA 0.1 mM, at 37jC. Activity was measuredthrough the decrease in the superoxide-dependent rate of cytochrome creduction. Results are the means F standard deviation of threeexperiments performed on different days. (B) Native polyacrylamidegel electrophoresis of CuZnSOD exposed to peroxynitrite. Preparationscontaining 0.3 Ag protein were loaded into a 15% native gel. The gelwas stained with NBT and superoxide dismutase activity is visible ascolorless spots. Lanes 1–5: 0, 0.05, 0.1, 0.4, and 1.5 mM peroxynitrite.

Fig. 2. pH dependence of the inactivation of CuZnSOD by peroxynitrite.CuZnSOD (5.8 AM) was exposed to peroxynitrite (1 mM) in sodiumphosphate buffer, 0.05 M, 0.1 mM DTPA, of varying pH. The opensymbols represent the activity of the samples incubated at the specifiedpH without peroxynitrite addition.

4.9 AM, a 50% decrease in activity was achieved with

a peroxynitrite concentration of f100 AM (IC50).

Similar inactivation profiles were obtained for bovine

CuZnSOD (not shown).

The inactivation was higher at alkaline pH (Fig. 2),

whereas preincubation of the enzyme alone at the

different pHs did not result in changes in activity.

Taking into account the fact that the natural substrate

of the enzyme is an anion, that the enzyme has a

positively charged anion channel, that the dismutase

activity does not change with pH within the range 5–

9 [43], and that in the case of the peroxidative

activity, the substrate is the HO2� anion, the fact that

the enzyme was preferentially inactivated at alkaline

pH can be due to peroxynitrite anion being the main

reactive species with CuZnSOD.

Kinetics

If peroxynitrite anion was the main reactive species,

it was likely that it was reacting directly with the

enzyme. In order to study the kinetics of the reaction,

an initial rate approach was used as before, because

pseudo-first-order concentrations of CuZnSOD with

respect to peroxynitrite cannot be achieved [28,40,44].

As observed in Fig. 3, the apparent rate constant

of peroxynitrite decay increased with CuZnSOD, con-

firming that a direct reaction occurs between the

enzyme and the peroxynitrite. From the slope of the

plot, a second-order rate constant of (9.4 F 1.0) � 103

Fig. 4. EPR spectra obtained from the reaction of peroxynitrite withCuZnSOD in the presence of MNP. Spectra A, peroxynitrite (1 mM) wasadded to CuZnSOD (0.5 mM monomer) in phosphate buffer (0.1 M,pH 7.4) containing DTPA (0.1 mM) and MNP (22 mM); B, CuZnSODalone; C, CuZnSOD and MNP in buffer; D, MNP alone in buffer; E,peroxynitrite was added to MNP; F, peroxynitrite was decomposed inbuffer containing MNP and CuZnSOD was added after 1 min (ROA,reverse order of addition control); G, same as F but CuZnSOD wasadded after 3 min; H, peroxynitrite was added to CuZnSOD in theabsence of MNP. Instrument settings were as follows: modulationamplitude, 5 G; time constant, 328 ms; scan time, 1.2 G/s; receivergain, 5 � 105; microwave power, 20 mW. Five scans were added toobtain the final spectrum. The scale of spectra C and D is 8-fold higherthan that of A, B, and E–H.

Fig. 3. Kinetics of peroxynitrite decay in the presence of CuZnSOD.Peroxynitrite (0.05 mM) was mixed with increasing concentrations ofCuZnSOD in 0.05 M sodium phosphate buffer, 0.1 mM DTPA, pH7.53, at 37jC. The absorbance at 302 nm was recorded for 10 s and theapparent rate constant of peroxynitrite decay was determined from theinitial slope as �(dA/dt)/(A0 � Al). The results are the means Fstandard deviation (n z 8) of a representative experiment.

Peroxynitrite inactivation of CuZnSOD 817

M�1 s�1 per monomer was calculated at pH 7.5 and

37jC.

EPR

Addition of peroxynitrite to CuZnSOD in the pres-

ence of the spin trap MNP led to detection of an

anisotropic signal, typical of an immobilized nitroxide

free radical protein adduct (Fig. 4, spectrum A).

Superimposed with the nitroxide spectrum was a

signal probably arising from copper, which was also

present in controls that contained CuZnSOD alone

(Fig. 4, spectrum B). When peroxynitrite was not

included in the reaction mixtures, the immobilized

nitroxide spectrum was replaced by a strong isotropic

three line signal with aN = 17.2 G (Fig. 4, spectrum

C). This signal is characteristic of di-t-butylnitroxide, a

contaminant which is frequently present in MNP

solutions. The di-t-butylnitroxide signal decreased in

reaction mixtures in which peroxynitrite was added,

suggesting that peroxynitrite or the radicals derived

from its homolysis were able to oxidize the di-t-

butylnitroxide to EPR-silent products (Fig. 4, spectra

A and C–E). When peroxynitrite was decomposed in

MNP-containing buffer previous to the addition of

CuZnSOD (reverse order of addition controls), the

immobilized nitroxide spectrum was not observed,

confirming that peroxynitrite was necessary for the

formation of the protein free radical (Fig. 4, spectra

F and G). Instead, a weak di-t-butylnitroxide signal

that increased with time was observed, suggesting that

the di-t-butylnitroxide adduct could be formed sponta-

neously after peroxynitrite had decayed. In the absence

of MNP, a different weak anisotropic signal was

observed after peroxynitrite reaction with CuZnSOD.

Fig. 5. EPR spectra obtained after proteolysis of peroxynitrite-treatedCuZnSOD. Spectrum A, peroxynitrite (1 mM) was added to CuZnSOD(0.5 mM monomer) in phosphate buffer (0.1 M, pH 7.4) containingDTPA (0.1 mM) and MNP (22 mM). After 1 min at room temperature,samples were gel filtered and Pronase (40 mg ml�1) and acetonitrile(14%) were added. Instrument settings: modulation amplitude, 1 G;time constant, 2.62 s; scan time, 6.71 G/s; receiver gain, 5 � 105;microwave power, 20 mW. Ten scans were added to obtain the finalspectrum. Spectrum B, computer simulation of spectrum A using thehyperfine coupling constants aN(NO) = 15.3 G, aN = 1.85 G, aN = 1.9 G,aH = 2.1 G, aH = 0.88 G [17]. The simulation program was allowed tosubtract the copper spectrum and to vary the linewidth for each linefrom the primary nitroxide. Spectrum C, peroxynitrite (7.1 mM) wasadded to L-histidine (100 mM) in phosphate buffer (0.1 M, pH 7.4)containing DTPA (0.1 mM) and MNP (22 mM). Instrument settings:modulation amplitude, 0.7 G; time constant, 1.31 s; scan time, 16.78G/s; receiver gain, 5 � 105; microwave power, 20 mW. One scan isshown. Spectrum D, computer simulation of spectrum C using thehyperfine coupling constants aN(NO) = 15.3 G, aN = 1.85 G, aN = 1.89G, aH = 2.1 G, aH = 0.8 G [17]. The spectrum of di-t-butylnitroxide(aN = 17.2 G) was included in the simulation, representing 11% of thetotal signal.

Table 1. Effects of Nitrite, Hydrogen Peroxide, Mannitol, and Bicarb-onate on Peroxynitrite-Dependent CuZnSOD Inactivation

Conditiona Superoxide

dismutase

activity (%)

No addition 100 F 11Peroxynitrite (1 mM) 16 F 5Reverse order of addition control (1 mM)b 91 F 8Peroxynitrite (1 mM) + nitrite (1 mM)c 47 F 7Peroxynitrite (1 mM) + nitrite (5 mM)c 65 F 9Nitrite (5 mM) 108 F 14Peroxynitrite (1 mM) + hydrogen peroxide (50 mM) 89 F 6Hydrogen peroxide (50 mM) 77 F 6Peroxynitrite (1 mM) + mannitol (100 mM) 54 F 9Peroxynitrite (1 mM) + sodium bicarbonate (25 mM) 51 F 6

a CuZnSOD (5 AM) was exposed to peroxynitrite in sodium

phosphate buffer (0.05 M, DTPA 0.1 mM, pH 7.4). After 3 min at

37jC, the samples were put on ice and diluted for activity measurement.b For the reverse order of addition control, peroxynitrite was

decomposed in buffer before the addition of CuZnSOD.c The amount of nitrite present as contaminant in the peroxynitrite

stock solution was 0.25 nitrite per peroxynitrite.

B. ALVAREZ et al.818

In order to attempt the assignment of the nitroxide

spectrum to a particular amino acid-derived radical,

reaction mixtures were subjected to nonspecific partial

proteolysis so as to increase the rotational dynamics of

the free radical adduct and improve the detection of

hyperfine couplings. Our attempts to mobilize the free

radical adduct were hampered by the relative instability

of the signal, by the spontaneous formation of di-t-

butylnitroxide, and by the fact that CuZnSOD is re-

markably resistant to proteolysis. Nevertheless, addition

of Pronase (40 mg ml�1) and acetonitrile (14%) to

peroxynitrite-treated CuZnSOD previously subjected to

gel filtration to remove excess MNP led to the conver-

sion of the anisotropic spectrum to a nearly isotropic

signal (Fig. 5, spectrum A). This spectrum showed a

complex pattern of hyperfine structure that suggested

couplings to several nitrogen and hydrogen atoms and

was highly similar to the spectra recently reported by

Gunther et al. for histidinyl radical formed from bovine

CuZnSOD exposed to hydrogen peroxide or peracetic

acid in the presence of MNP [17]. Accordingly, expo-

sure of free L-histidine to peroxynitrite led to the

detection of a signal similar to that reported from the

incubation of histidine with Fenton reagent and MNP

by Gunther et al. (Fig. 5, spectrum C), which was

assigned, based on 13C labeling, to the histidinyl radical

centered in the C-2 of the imidazole ring [17]. Taken

together, our EPR experiments are consistent with the

formation of a histidinyl radical in human CuZnSOD

exposed to peroxynitrite.

Effects of nitrite, hydrogen peroxide, mannitol, and

bicarbonate on the inactivation

In order to observe the inactivation of CuZnSOD by

peroxynitrite, it was important to avoid high concentra-

tions of nitrite in the reaction mixtures. Nitrite is a usual

contaminant in peroxynitrite preparations, both as

unreacted synthesis reagent and as a decomposition

product. As observed in Table 1, nitrite protected CuZn-

SOD from inactivation. To prevent possible artifacts, we

checked the effect of nitrite on the activity assay. At

millimolar concentrations, nitrite interfered with the

activity assay. For instance, 4.5 mM nitrite decreased

the rate of cytochrome c reduction by 7% (data not

shown), possibly through the xanthine oxidase-depen-

dent reduction of nitrite to nitric oxide, which competes

for superoxide [45]. However, the amounts of nitrite that

were carried from the SOD/peroxynitrite reaction mix-

tures into the activity assays were 0.5–2 and 1.2–5 AMfor the 1 and 5 mM nitrite conditions. At these concen-

trations, no interference with the rate of cytochrome c

reduction was observed (data not shown), in agreement

Peroxynitrite inactivation of CuZnSOD 819

with the values of KM for nitrite of xanthine oxidase

(15–40 mM [45]). Thus, it can be concluded that nitrite

indeed prevented the inactivation of CuZnSOD by per-

oxynitrite. So, care was taken throughout this work to use

peroxynitrite preparations that had less than 0.3 nitrite

per peroxynitrite, and nitrite concentration was measured

in each experiment.

Hydrogen peroxide inactivates CuZnSOD and is an-

other usual contaminant in peroxynitrite solutions. Thus,

we assessed its effect on the peroxynitrite-dependent

inactivation. When peroxynitrite was decomposed in

the buffer previous to CuZnSOD addition (reverse order

of addition control), no inactivation was observed, ruling

out a role for contaminating hydrogen peroxide in the

inactivation (Table 1). Moreover, hydrogen peroxide (50

mM) protected CuZnSOD from peroxynitrite. In turn,

hydrogen peroxide alone inactivated the enzyme by

f25% in the 3 min that the experiment lasted, in

agreement with the relatively slow rate of inactivation

at pH 7.4 [46].

In order to assess if the hydroxyl radical derived from

peroxynitrite homolysis had a role in the inactivation of

the enzyme, we evaluated the effect of mannitol, a

classical hydroxyl radical scavenger big enough not to

enter the active site. Mannitol partially protected the

enzyme from inactivation, suggesting that, in addition to

the direct reaction of peroxynitrite anion, hydroxyl radical

can also be implicated in the inactivation (Table 1).

Peroxynitrite reacts with carbon dioxide, and this

reaction is relevant in vivo due to the relatively high

concentration of carbon dioxide in biological systems

(1–2 mM). To investigate its effect, we added sodium

bicarbonate (25 mM) to the buffer, so that the concen-

tration of carbon dioxide was 1.2 mM at pH 7.4 accord-

ing to the pKA of 6.1 for the pair bicarbonate/carbon

dioxide. Carbon dioxide partially protected CuZnSOD

from peroxynitrite inactivation (Table 1).

Scheme 1. Reaction of CuZnSODwith peroxynitrite. Peroxynitrite anioncan react with CuZnSOD with a rate constant of 9.4 � 103 M�1 s�1,leading to the formation of a copper-bound oxidizing species that reactswith histidine, forming histidinyl radical. Alternatively, peroxynitritecan homolyze to hydroxyl and nitrogen dioxide radicals or, in thepresence of carbon dioxide, to carbonate radical and nitrogen dioxide.

DISCUSSION

Peroxynitrite leads to human CuZnSOD inactivation

and histidinyl radical formation. With an IC50 of 100 AMat 4.9 AM CuZnSOD, the enzyme is moderately sensitive

toward peroxynitrite. It is less sensitive than MnSOD,

whose IC50 is 10 AM at 0.5 AM enzyme [26], but it is

comparable to another metalloprotein such as tyrosine

hydroxylase, whose IC50 is 100–200 AM at 18 AMenzyme [44]. Although CuZnSOD shows moderate sen-

sitivity toward peroxynitrite, the inactivation of CuZn-

SOD with peroxynitrite may become more relevant in

light of the possible in situ formation of peroxynitrite

from the reaction of dioxygen with reduced, zinc-deplet-

ed enzyme in the presence of nitric oxide [20]. Perox-

ynitrite reacted directly with CuZnSOD with a second-

order rate constant of (9.4 F 1.0) � 103 M�1 s�1 per

monomer. This value is lower than that reported for

MnSOD (2.5 � 104 M�1 s�1 per monomer) [28], in

agreement with the fact that MnSOD is more sensitive to

peroxynitrite than CuZnSOD.

Taking into account, together with our results, (a) that

the inactivation of CuZnSOD mediated by hydrogen

peroxide involves the formation of a copper-bound

hydroxyl radical species and (b) that the nitration of

phenolics by peroxynitrite is promoted by CuZnSOD, it

can be proposed that peroxynitrite anion reacts with

Cu(II) in the active site, forming nitrogen dioxide and

an oxidizing species, probably hydroxyl radical bound to

the copper (Scheme 1). This species is likely to be

responsible for the formation of histidinyl radical by

reacting with histidine residues in the active site, in line

with what has been proposed for hydrogen peroxide. In

turn, the formation of histidinyl radical also constitutes

evidence for the existence of free radical mechanisms in

the CuZnSOD-promoted nitration of phenolics, as op-

posed to the mechanism proposed involving nitronium

cation (NO2+) [23,24]. Further reaction of histidinyl

radical with oxygen has been reported to lead to histi-

dine peroxyl radical and to 2-oxohistidine [17]. In the

context of the simultaneous formation of nitrogen diox-

ide, histidinyl radical could also lead to nitrohistidine.

The possibility of nitrohistidine formation has been

observed for histidine-containing peptides (GHG and

GHK), exposure of which to peroxynitrite led to the

mass spectrometric detection of derivatives with a mass

increase of +45, consistent with the formation of nitro-

histidine and confirmed through MS/MS of the parent

ions (B. Alvarez et al., unpublished observations).

Whereas the detection of histidinyl radical from the

reaction of CuZnSOD with peroxynitrite and hydrogen

B. ALVAREZ et al.820

peroxide [17] is consistent with the site-specific oxida-

tion of histidines, the formation of tryptophanyl radical

at the protein surface has been reported under conditions

in which the long-lived and selective carbonate radical

was formed [47].

The effects of nitrite, hydrogen peroxide, mannitol,

and bicarbonate can be accounted for by the known

biochemistry of peroxynitrite and CuZnSOD. In the case

of nitrite, whose direct reaction with peroxynitrite is

negligible [48], the fact that it prevented the inactivation

of the enzyme can be interpreted by competition between

the two anions for the active site or by nitrite scavenging

of the reactive species bound to the active site. Regarding

the reaction between CuZnSOD and hydrogen peroxide,

it has been shown that nitrite can inhibit the hydrogen

peroxide-mediated inactivation of CuZnSOD, with 50

mM nitrite completely preventing the inactivation, being

in turn oxidized toSNO2, and promoting the formation of

nitrotyrosine [8,12–14,49]. In this same line, it seems

likely that, in the case of peroxynitrite, nitrite reacted

with the active site-bound oxidant to form nitrogen

dioxide, which is only a moderate one-electron oxidant

(EjV[SNO2/NO2�] = 0.99 V) [50]. The fact that hydrogen

peroxide prevented the inactivation of CuZnSOD can

also be interpreted in terms of competition or scavenging

of the active site-bound oxidant because, again, hydrogen

peroxide does not react directly with peroxynitrite

[51,52]. The reaction of hydrogen peroxide with the

copper-bound hydroxyl radical would lead to superoxide

radical, which is innocuous toward CuZnSOD. In con-

trast, the effect of bicarbonate is consistent with the fact

that carbonate radical is able to inactivate the enzyme.

Carbon dioxide does react directly with peroxynitrite,

with a rate constant of 4.6 � 104 M�1 s�1 at pH 7.4 and

37jC [53], leading to carbonate radical in f35% yield.

Under our conditions, we can calculate with Gepasi from

the known rate constants and concentrations that more

than 95% of peroxynitrite reacted initially with carbon

dioxide. Thus, the fact that the protection of peroxynitrite

inactivation by carbon dioxide was not complete but the

enzyme was nevertheless 50% inactivated in its presence

suggests that the carbonate radical was able to react with

CuZnSOD, leading to its inactivation, in line with

carbonate radical being a strong one-electron oxidant

(EjV[CO3S�, H+/HCO3

�] = 1.78 V) [54]. Our results

agree with those reported by Yamakura et al. [55], who

observed an f30% decrease in the activity of CuZnSOD

exposed to peroxynitrite in the presence of carbon

dioxide, and are consistent with the effect of bicarbonate

on the inactivation of CuZnSOD by hydrogen peroxide,

in the sense that the carbonate radical formed from the

reaction of the copper-bound hydroxyl radical with

carbon dioxide was able to inactivate the enzyme

[13,14,16,46]. Analogously, the partial protection exerted

by mannitol, which does not react directly with perox-

ynitrite [52], can be rationalized by the fact that hydroxyl

radical was also able to inactivate the enzyme. Although

peroxynitrite can react directly with the enzyme, under

the experimental conditions used, i.e., 5 AM enzyme and

1 mM peroxynitrite, it can be calculated that more than

99% of peroxynitrite isomerized to nitrate via homolysis,

with free nitrogen dioxide and hydroxyl radicals formed

in f30% yield. Hydroxyl radical is so reactive that it is

likely to react at the surface of the protein. However,

considering that about 20 hydroxyl radical collisions are

needed for a CuZnSOD inactivation event [56,57], a

significant amount of the enzyme may have been inacti-

vated under our conditions, under which the concentra-

tion of hydroxyl radical formed was relatively high

(f0.3 mM). Alternatively, secondary species formed

from hydroxyl radical reaction with amino acids in the

surface of the protein may have been involved in the

inactivation, and the partial protection exerted by man-

nitol may be due to the inhibition of the formation of

these species.

The inactivation of CuZnSOD with peroxynitrite is

summarized in Scheme 1. The fact that CuZnSOD is

inactivated by peroxynitrite is relevant because a de-

crease in the dismutase activity may lead to the ampli-

fication of oxidative damage, and a slowing in the rate of

superoxide dismutation may further increase the forma-

tion of peroxynitrite through its reaction with nitric

oxide. Concerning ALS, although evidence points to a

toxic gain of function of CuZnSOD instead of a loss of

dismutase activity as responsible for the onset of the

disease, inactivation could be a contributing factor.

Indeed, markers of oxidative damage have been found

increased in both sporadic and familial forms of the

disease [58–60], and CuZnSOD has been identified as

one of the oxidized targets [58]. Hopefully, our results

will help us to better understand the interactions between

nitric oxide-derived species and CuZnSOD in ALS.

Acknowledgments—This work was supported by CSIC (Universidadde la Republica, Uruguay) and The Third World Academy of Sciences(to B. Alvarez), The Howard Hughes Medical Institute, theGuggenheim Foundation, and Fogarty-NIH (to R. Radi). We thankDrs. Mario Senorale, Gerardo Ferrer, and Celia Quijano for helpfuldiscussions.

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ABBREVIATIONS

ALS—amyotrophic lateral sclerosis

CuZnSOD—copper–zinc superoxide dismutase

DTPA—diethylenetriaminepentaacetic acid

EPR—electron paramagnetic resonance

MNP—2-methyl-2-nitrosopropane

NBT—4-nitroblue tetrazolium

TEMED—N,N,NVNV-tetramethylethylenediamine