Archives of Biochemistry and Biophysics 421 (2004) 99–107

ABBwww.elsevier.com/locate/yabbi

Cytochrome c: a catalyst and target of nitrite-hydrogenperoxide-dependent protein nitration

Laura Castro,a,b,c,* Jason P. Eiserich,a,b,d Scott Sweeney,a,b Rafael Radi,c

and Bruce A. Freemana,b,e

a Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL 35233, USAb The Center of Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35233, USA

c Department of Biochemistry and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Rep�uublica,Montevideo 11800, Uruguay

d Division of Nephrology, Department of Internal Medicine, University of California at Davis, Davis, CA 95616, USAe Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35233, USA

Received 6 June 2003, and in revised form 18 August 2003

Abstract

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions

of nitric oxide (�NO), with peroxynitrite (ONOO�) and leukocyte peroxidase-derived nitrogen dioxide (�NO2) being proximal

mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on

the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO�2 ) and hydrogen

peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid,

Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and �NO2 production, as for

phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic

fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as

catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive ty-

rosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and

NO�2 , with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that

is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the

distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to

biomolecule nitration reactions.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Cytochrome c; Mitochondria; Peroxidase; Manganese superoxide dismutase; Nitric oxide; Nitrite; Peroxynitrite; Hydrogen peroxide

Post-translational nitration of tyrosine to yield 3-ni-trotyrosine (NO2Tyr)

1 has been detected in various or-

gans and cell types both clinically and in animal models

* Corresponding author. Fax: +598-2-924-9563.

E-mail address: lcastro@fmed.edu.uy (L. Castro).1 Abbreviations used: �NO, nitric oxide; ONOO�, peroxynitrite;

H2O2, hydrogen peroxide, NO2Tyr, nitrotyrosine, ABTS, 2-20-azino-

bis (3-ethylbenzthiazoline-6-sulfonic acid), DTPA, diethylenetriamine-

pentaacetic acid; Mn-SOD, manganese superoxide dismutase; HPA,

para-hydroxyphenylacetic acid; BSA, bovine serum albumin, HPLC,

high-pressure liquid chromatography.

0003-9861/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2003.08.033

of acute and chronic inflammation [1,2]. In addition tonitration of free tyrosine, a number of proteins are

modified by nitration of specific tyrosine residues, in-

cluding tyrosine hydroxylase, sarcoplasmic reticulum

Ca2þ-ATPase, prostacyclin synthase, neurofilaments,

a-synuclein, and Mn-superoxide dismutase (Mn-SOD).

The magnitude of protein tyrosine nitration during in-

flammatory processes that involve accelerated rates of

reactive oxygen species and �NO production ranges from0.01 to 0.1mol%, quantitatively similar to tyrosine

phosphorylation. Incorporation of this bulky derivative

into tyrosine can induce alterations to tyrosine-mediated

100 L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107

electron transfer reactions, as well as protein structureand function [3–7].

Addition of a nitro group (–NO2) to the ortho posi-

tion of tyrosine can occur via multiple �NO-dependent

mechanisms, with �NO2 frequently being the proximal

mediator of tyrosine nitration in tissues. For instance,

ONOO� and its conjugate acid, peroxynitrous acid

(ONOOH) (pka ¼ 6:8), yield �NO2via homolytic cleav-

age. In biological milieu, reaction of ONOO� withcarbon dioxide (CO2) predominates, due to both the

high CO2 concentrations typically present in the context

of other competing ONOO� targets and the fast reaction

of CO2 with ONOO� (k ¼ 5:8� 104 M�1 s�1 [8]). The

product of ONOO� reaction with CO2, ONOOCO�2

(nitrosoperoxocarboxylate), homolyzes to yield species

CO��3 (carbonate radical) and �NO2, [9,10] that will

readily yield �Tyr (tyrosyl radical) and the subsequentproduction of NO2Tyr. An increase in CO2 concentra-

tion, especially from the hypercapnic conditions often

experienced during inflammation and disruption of tis-

sue metabolic homeostasis, will accelerate this pathway

[11]. In addition, ONOO�-dependent nitration is en-

hanced by metal center catalysis of the formation of a

nitronium-like intermediate (NOþ2 , [9]).

In many inflammatory diseases, NO�2 accumulates to

concentration ranging from 10 to 50 lM in plasma and

alveolar lining fluids following induction of �NO syn-

thases, with NO�2 concentrations often higher in tissue

microenvironments than in plasma [12,13]. Heme pro-

tein peroxidases, such as neutrophil myeloperoxidase

(MPO) and eosinophil peroxidase (EPO), react with

H2O2 to form compound I, which readily generates�NO2 via NO�

2 one-electron oxidation [14–17].Mitochondria are the principal intracellular loci of

H2O2 production in non-phagocytic cells. Under basal

conditions, mitochondrial H2O2 generation represents

up to 2% of the oxygen consumption by this organelle,

with mitochondrial H2O2 fluxes increased by drugs or

toxins such as electron transport inhibitors, uncouplers,

redox cycling molecules, exposure to hyperoxia, and

during ischemia–reperfusion events [18–21]. Mito-chondrial proteins are also important targets of reac-

tive oxygen and nitrogen species. For instance, �NO

inhibits mitochondrial electron transport via reversible

binding to cytochrome oxidase and ONOO� inacti-

vates complex I, II, and ATPase [22–26]. Also, gener-

ation of nitrating species inactivates matrix-localized

Mn-SOD via nitration of Tyr34, a phenomenon

observed during human kidney allograft rejection[7,27,28].

Cytochrome c catalyzes the H2O2-mediated oxidation

of various electron donors including ABTS, 4-amino-

antipyrine, and luminol, and participates as a catalyst in

mitochondrial lipid peroxidation. Thus, it has been

proposed that hemoproteins with peroxidase-like activ-

ity contribute to mitochondrial oxidative damage

[29–31]. This globular heme protein of 13 kDa is local-ized on the intermembrane side of the inner mitochon-

drial membrane and participates in respiration,

transferring electrons between complex III and IV

[32,33]. Upon release from mitochondria during a vari-

ety of metabolic and toxic insults, cytochrome c par-

ticipates in the signaling pathways underlying apoptotic

cell death by binding to APAF-1 (apoptotic protease

activating factor-1) and triggering activation of procas-pase 9 in a complex called the apoptosome [34]. Cyto-

chrome c is also a target for reactive nitrogen species. In

particular, ONOO� reacts rapidly and oxidizes (k ¼ 2�105 M�1 s�1) cytochrome c2þ and promotes the nitration

of Tyr67 in cytochrome c3þ. Nitration of Tyr67 induces

profound changes in the redox properties of cytochrome

c, including an increase in peroxidase activity [35,36]. In

addition, nitrated cytochrome c was detected in vivofrom rat kidneys from allograft nephropathy [37].

Herein, we report that cytochrome c, in the presence

of biologically relevant NO�2 and H2O2 concentrations,

serves as a catalyst for self-nitration and nitration of

proximal phenolic molecules and protein tyrosines. The

reaction mechanisms leading to NO2Tyr formation by

this pathway involve heme peroxidase-like formation of�Tyr and �NO2. These data reveal mechanism adding tothe redundancy of pathways leading to tyrosine nitra-

tion and support the significance of this post-transla-

tional protein modification in processes of cell injury

and signaling.

Materials and methods

Reagents

Horse heart cytochrome c (C-7752), microperoxidase-

11 (MPx-11), potassium phosphate (mono and dibasic),

diethylenetriaminepentaacetic acid (DTPA), nitrite, hy-

drogen peroxide (H2O2), manganese dioxide (MnO2),

xanthine, sodium bicarbonate, bovine serum albumin

(BSA), Tween 20, sodium dithionite, 4-nitroblue tetra-zolium, riboflavin, N,N,N 0,N 0-tetramethylethylenedia-

mide, and 2-20-azino-bis (3-ethylbenzthiazoline-6-sulfonicacid) (ABTS) were purchased from Sigma Chemical (St.

Louis, MO, USA). Potassium cyanide and para-hy-

droxyphenylacetic acid (HPA) were from Aldrich (Mil-

waukee, WI, USA). Porcine pancreas trypsin was from

Gibco Life Technologies (Rockville, MD, USA). Bovine

milk xanthine oxidase was obtained from Calbiochem–Novabiochem (La Jolla CA, USA). Recombinant hu-

man Mn-SOD was a gift from Prof. Claude Piantadosi

(Duke University). Peroxynitrite was synthesized in a

quenched-flow reactor as previously described [38] and

excess H2O2 was removed by treatment with MnO2.

Peroxynitrite concentrations were determined spectro-

photometrically at 302 nm (�302 ¼ 1670M�1 cm�1) [38].

L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107 101

Cytochrome c catalyzed nitration

Cytochrome c3þ (200 lM) was incubated for 30min

with NO�2 (0–500 lM) and H2O2 (3.33 nmol/min, total

H2O2 accumulated, 500 lM) delivered using a motor-

driven syringe pump in 100mM potassium phosphate

buffer, pH 7.4, including 100 lM DTPA (total volume

200 ll) at 25 �C. In some experiments, Mn-SOD (45 lM)

or BSA (2.4 lM) was included. Aliquots were withdrawnfor Western immunoblot analysis or SOD activity de-

termination.

SOD activity

SOD activity was determined via inhibition of cyto-

chrome c3þ reduction by superoxide (O��2 ) generated by

xanthine plus xanthine oxidase and by inhibition of ni-troblue tetrazolium oxidation in polyacrylamide activity

gels [39].

Isolation of rat liver mitochondria and mitoplast prepa-

ration

Intact rat liver mitochondria were prepared by dif-

ferential centrifugation as previously described [40].Mitochondrial pellets were resuspended in a minimal

volume of homogenization buffer (0.3M sucrose, 5mM

potassium phosphate, 1mM EGTA, and 0.1% bovine

serum albumin, pH 7.4), protein concentration was de-

termined according to the Bradford method, and the

respiratory control ratio was measured for complex II

using a Clark-type oxygen electrode. Cytochrome c-de-

pleted mitoplasts were prepared as described previously[41]. Briefly, mitochondria were resuspended in 10mM

KCl, 2mM Tris–HCl, pH 7.4, at 2mg/ml, incubated for

10min at 37 �C, and centrifuged at 10,000g for 10min at

4 �C. Pellets were resuspended in 150mM KCl, 2mM

Tris–HCl, pH 7.4, to extract cytochrome c. The proce-

dure was repeated twice and cytochrome c extraction

was confirmed after addition of dithionite by measuring

the absorbance at 550 nm in supernatants. Typically,0.6� 0.1 nmol cytochrome c/mg mitochondrial protein

was extracted.

Western blot analysis

SDS–PAGE of protein samples was performed on

15% polyacrylamide gels and proteins were transferred

electrophoretically (15V, 1 h, semi-dry system) to PVDFmembranes (Immobilon P-Millipore). Membranes were

blocked with 5% bovine serum albumin (BSA) in 50mM

Tris–chloride, pH 7.4, 150mM NaCl (TBS), and 0.3%

Tween 20 (blocking buffer). For detection of NO2Tyr,

PVDF membranes were incubated (1 h at 25 �C) with

1 lg/ml anti-nitrotyrosine polyclonal antibody (1/1000

dilution, a gift from Dr. Joe Beckman, Oregon State

University) in blocking buffer. For cytochrome c de-tection, membranes were incubated with 2 lg/ml

monoclonal anti-cytochrome c antibody (1/500 dilution,

clone 7H8.2C12) from Pharmingen. After extensive

washing in TBS and 0.3% Tween 20, the immunocom-

plexed membranes were probed (1 h at 25 �C) with

horseradish peroxidase-linked secondary antibody (1/

300,000 dilution) in TBS, 5% BSA, and 0.3% Tween 20.

Probed membranes were washed with TBS, 0.3% Tween20 and immunoreactive proteins were visualized by lu-

minol-enhanced chemiluminescence (Pierce, Rockford,

IL, USA).

High-performance liquid chromatography analysis of

HPA oxidation and nitration products

Different concentrations of HPA were incubated for30min at 25 �C with either 20 lM cytochrome c3þ or

MPx-11, 500 lM NO�2 and 3.33mol/min H2O2 were

delivered using a motor-driven syringe pump in 100mM

potassium phosphate, pH 7.4, plus 100 lM DTPA in a

total volume of 200 ll. Cytochrome c was immediately

removed by microfiltration using 5000 molecular weight

cutoff filters (Fisher, Pittsburgh, PA, USA). HPA and its

products were analyzed by reverse-phase high-perfor-mance liquid chromatography (HPLC) on a 5 lmSpherisorb ODS-2 column (4mm� 25 cm), via isocratic

elution with 30% methanol in 100mM KH2PO4, pH 3.0,

for 25min. Products were identified by UV detection

(274 nm) and quantified by use of external standards. Bi-

HPA was specifically identified via an in-line fluores-

cence detection at kex ¼ 284 nm, kem ¼ 410 nm.

Cell lysate preparation, incubation with cytochrome c, and

peroxidase activity measurements

Jurkat cells were grown in RPMI and 10% fetal bo-

vine serum at 37 �C in a 5% CO2 atmosphere. Cells

(1� 0.2� 106 cells/ml) were harvested, washed twice

with PBS, pelleted, and resuspended in 400 ll lysis buffer(20mM Hepes–KOH, pH 7.0, 10mM KCl, 1.5mMMgCl2, 0.1mM CaCl2, and 250mM sucrose). Cells were

kept on ice for 15min and then sonicated. Cell lysates

were stored at )70 �C until use. Cell lysates were freeze–

thawed twice, insoluble material was removed by

centrifugation at 10,000g for 10min prior to use, and

protein content was determined. Cytochrome c3þ

(62.5 lM) was incubated with 7mg/ml cell lysate protein

in 100mM sodium phosphate, pH 7.4, at 37 �C for 4 h.In some cases, the pH was adjusted to 5 prior to incu-

bation. As a control, cytochrome c was incubated in

buffer without cell lysate proteins or with 1 and 10 lg/ml

trypsin. Aliquots were then removed and peroxidase

activity was determined or incubated with Mn-SOD in

the presence of NO�2 /H2O2 as previously described,

followed by Western blot analysis of protein nitration.

102 L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107

Peroxidase activity was measured spectrophotometri-cally, following the oxidation of ABTS at 420 nm

(k ¼ 3:6� 104 M�1 cm�1) as previously [29].

General conditions

Experiments reported herein were performed a min-

imum of three times with similar results being obtained.

Results are expressed as means� SD or by a represen-tative example. Graphics were generated in Slide-Write

5.0 for Windows (Advanced Graphic Software).

Fig. 2. Effect of nitrite on the H2O2-dependent loss of the Soret band

of cytochrome c. The Soret band of cytochrome c3þ (12.5lM) was

followed at 408 nm during the infusion of 3.33 nmol/min H2O2 for

30min, in 100mM potassium phosphate, pH 7.4, plus 100lM DTPA

at 25 �C while stirring using a Hellma cuv-o-stirr model 333. Condi-

tions were: (A) control (without H2O2); (B) H2O2 to cytochrome c3þ

pre-incubated with 5mM KCN for 30min; (C) H2O2 plus 500lMNO�

2 ; and (D) H2O2 only.

Results

Cytochrome c nitration by H2O2 +NO�2

Cytochrome c3þ (200 lM) was incubated with NO�2

(0–500 lM) in the presence of H2O2 (3.33 nmol/min,

total H2O2 accumulated, 500 lM) for 30min or exposed

to bolus addition of ONOO� (0–1mM) and examined

by Western blot analysis for cytochrome c or nitroty-

rosine immunoreactivity. Both NO�2 +H2O2 and

ONOO� catalyzed cytochrome c3þ nitration (Fig. 1).

When NO�2 concentrations increased from 0 to 50 lM,

trimeric and tetrameric forms of cytochrome c were

formed, diminishing at higher NO�2 concentrations

when NO2Tyr immunoreactivity became more promi-

nent (Fig. 1). Whereas 10mM methionine completely

abolished ONOO�-dependent cytochrome c nitration, it

did not change cytochrome c nitration yields induced by

NO�2 +H2O2 (not shown).

Incubation of cytochrome c3þ (12.5 lM) with3.33 nmol/min H2O2 led to a progressive loss of the

Soret absortion band, which can be followed at 408 nm.

The addition of 500 lM NO�2 delayed the decay at

408 nm while preincubation with 5mM KCN com-

pletely abolished the heme bleaching induced by H2O2

(Fig. 2).

Fig. 1. Cytochrome c nitration. Cytochrome c3þ (200lM) was exposed to ox

25 �C by either a bolus addition of ONOO� or different NO�2 concentrations a

Five micrograms of each sample was run on a SDS–15% PAGE and examined

Cytochrome c catalyzes nitration of other proteins

In the presence of NO�2 , cytochrome c+H2O2 in-

duced nitration of tyrosine residues in other proteins.

Reaction of 200 lM cytochrome c3þ with 2.4 lMBSA in

the presence of 0.5mM NO�2 and 3.33 nmol/min H2O2

resulted in the nitration of both cytochrome c and BSA

tyrosine residues, as detected by Western blottingagainst NO2Tyr (Fig. 3).

Microperoxidase-11 was utilized as a model cyto-

chrome c heme. Microperoxidases are obtained by di-

gestion of cytochrome c with proteolytic enzymes and

consist of ferriheme c covalently linked to a peptide

chain of varying lengths that includes His18, generally

assumed to be axially ligated to the heme iron but lacks

the sixth coordination position bound to Met80 incytochrome c [42]. It has been recently shown that

idants in 100mM potassium phosphate, pH 7.4, plus 100 lM DTPA at

nd 3.33 nmol/min H2O2 for 30min (total H2O2 accumulated, 500lM).

by Western Blot analysis against anti-cytochrome c and anti-NO2Tyr.

Fig. 3. Cytochrome c catalyzes nitration and oxidation of BSA. Bovine

serum albumin (2.43lM) was incubated with cytochrome c3þ or MPx-

11 (200 lM) in 100mM potassium phosphate, pH 7.4, plus 100lMDTPA, at 25 �C and exposed to 500lM NO�

2 plus 3.33 nmol/min

H2O2 for 30min. Twenty micrograms of each sample was separated on

SDS–15% PAGE and examined by Western blot analysis against anti-

NO2Tyr. Where indicated, cytochrome c3þ or MPx-11 was pre-incu-

bated with 5mM KCN for 30min.

L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107 103

microperoxidase-8 is able to catalyze the nitration of

phenolic compunds by NO�2 in the presence of H2O2

[43]. Greater yields of BSA nitration were observed

when incubated with 200 lM MPx-11, 0.5mM NO�2 ,

Fig. 4. Cytochrome c catalyzes nitration and oxidation of Mn-SOD but doe

(45lM) was added to cytochrome c3þ or MPx-11 (200lM) in 100mM potass

to 500lM NO�2 plus 3.33 nmol/min H2O2 for 30min. Where indicated, 25

separated on SDS–15% PAGE and examined by Western blot analysis agains

native PAGE separation was performed, and the gel was developed for SOD

Corresponding SOD activity was determined spectrophotometrically via inh

dase.

and 3.33 nmol/min H2O2, compared to cytochromec under similar conditions (Fig. 3). Nitration of con-

taminant cytochrome c and high molecular weight

aggregates of nitrated proteins that were not reducible

by b-mercaptoethanol were also observed under these

conditions (Fig. 3).

Preincubation of MPx-11 or cytochrome c3þ with

5mM KCN for 30min completely abolished BSA ni-

tration, with increased dimerization of cytochrome c

concomitantly observed (Fig. 3). Reaction of either

200 lM cytochrome c3þ or MPx-11 with 11 lM human

Mn-SOD, in the presence of 0.5mM NO�2 and

3.33 nmol/min H2O2, resulted in Mn-SOD tyrosine ni-

tration and appearance of high molecular weight protein

aggregates, as detected by Western blotting against

NO2Tyr (Fig. 4A). Again, the extent of Mn-SOD ni-

tration was greater when catalyzed by MPx-11, com-pared with cytochrome c. No tyrosine nitration was

observed when Mn-SOD was incubated with NO�2 and

H2O2 alone (Fig. 4A). Addition of 25mM HCO�3 (in

equilibrium with 1.3mM CO2) did not increase cyto-

chrome c or Mn-SOD nitration (Fig. 4A).

Mn-SOD contains nine tyrosines, with the nitration

of Tyr34 located only a few angstroms from the active

site manganese responsible for the inactivation of hu-man Mn-SOD by ONOO� [28]. Thus, the fast reaction

between Mn-SOD and ONOO� (k ¼ 1� 105 M�1 s�1)

leads to Tyr34 nitration and inactivation of Mn-SOD

[27,28,44]. Nitration of Mn-SOD catalyzed by cyto-

chrome c/H2O2/NO�2 or MPx-11/H2O2/NO�

2 did not

induce significant enzyme inactivation. This was

s not inhibit enzyme catalytic activity. Recombinant human Mn-SOD

ium phosphate, pH 7.4, plus 100lM DTPA, at 25 �C and then exposed

mM sodium bicarbonate was added. In (A), 5 lg of each sample was

t NO2Tyr. In (B), 250 ng of each sample was applied in each lane, 10%

activity. Peroxynitrite-treated (0.5mM) Mn-SOD was also evaluated.

ibition of reduction of cytochrome c3þ by xanthine plus xanthine oxi-

Fig. 6. Cytochrome c catalyzes nitration and oxidation of hydroxy-

phenylacetic acid. Different concentrations of HPA were incubated3þ

104 L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107

affirmed by both activity gel analysis and by measuringthe inhibition of O:�

2 -dependent reduction of cyto-

chrome c3þ (Fig. 4B), even though nitration occurred

that was extensive enough to induce increased migration

toward the anode in MPx-11-treated Mn-SOD activity

gels (Figs. 4A and B). The change in the electrophoretic

properties of nitrated Mn-SOD species infers a de-

creased isoelectric point compatible with the nitration of

tyrosine residues and a consequent lowering of the pKa.As previously, ONOO�-treated Mn-SOD resulted in

both nitration and enzyme inactivation (Figs. 4A and B,

[27,28,44].

To explore the role of cytochrome c in catalyzing

NO�2 plus H2O2-dependent nitration of mitochondrial

proteins, responses of cytochrome c-depleted mitoplasts

were compared with those of cytochrome c-supple-

mented rat liver mitoplasts (Fig. 5). Re-supplementationof rat liver mitoplasts with cytochrome c resulted in

significantly greater extents of protein nitration (Fig. 5),

supporting a role for cytochrome c in mitochondrial

protein nitration during oxidative stress.

Cytochrome c catalyzed nitration and oxidation of HPA

The reaction of cytochrome c3þ or MPx-11 andH2O2 +NO�

2 showed a dose-dependent increase of ni-

tro-hydroxyphenylacetic acid (NO2HPA) generation at

with either 20 lM cytochrome c (A) or MPx-11 (B) and 500lMNO�

2 plus 3.33 nmol/min H2O2 for 30min in 100mM potassium

phosphate, pH 7.4, plus 100lM DTPA, at 25 �C. HPA and its prod-

ucts were quantified by reverse-phase HPLC.

Fig. 5. Depletion of cytochrome c reduces nitration in mitoplasts ex-

posed to NO�2 and H2O2. Cytochrome c-depleted (lane 2) or cyto-

chrome c-depleted/repleted (lane 3) mitoplasts were exposed to 500lMNO�

2 plus 3.33 nmol/min H2O2 for 30min in 100mM potassium

phosphate, pH 7.4, plus 100lM DTPA, at 25 �C. Fifty micrograms of

each control (lane 1) or treated (lanes 2 and 3) sample was separated on

SDS–15% PAGE and examined by Western blot analysis against anti-

NO2Tyr.

HPA concentrations less than 0.2mM. Similar maxi-

mum yields of NO2HPA were obtained with either cy-tochrome c or MPx-11. At greater HPA concentrations,

there was a dose-dependent decrease in NO2HPA for-

mation in concert with increased bi-hydroxyphenylace-

tic acid (biHPA) generation (Fig. 6). Preincubation of

MPx-11 or cytochrome c3þ with 5mM KCN for 30min

completely inhibited H2O2-dependent NO2HPA for-

mation (not shown) indicating that the nitration and

oxidation reactions were heme-dependent. No NO2HPAwas observed when HPA was incubated with H2O2 and

NO�2 in the absence of cytochrome c or MPx-11 (not

shown), excluding artifictual nitration due to an acidic

HPLC mobile phase.

Partial proteolysis of cytochrome c increases cytochrome

c peroxidase activity and Mn-SOD nitration

MPx-11 catalyzed greater nitration yields than cyto-

chrome c in proteins (Figs. 3 and 4). Also, MPx-11

displayed �520 times greater peroxidase activity to-

wards ABTS than cytochrome c, when equimolar con-

centrations of MPx-11 and cytochrome c were

compared. To test the concept that partial proteolysis

Fig. 7. Proteolysis of cytochrome c increases both cytochrome c per-

oxidase and nitrating activities. Cytochrome c (15 lM) was added to

Mn-SOD as the native protein or following treatment with Jurkat cell

lysates or trypsin for 2 h at 37 �C, all prior to addition of NO�2 and

H2O2. Lane 1 represents protein (cytochrome c3þ, 15 lM and Mn-

SOD, 24 lM) without addition of NO�2 and H2O2; lane 2, native cy-

tochrome c; lane 3, cytochrome c treated with Jurkat cell lysate at pH

7.4; lane 4, cytochrome c treated with Jurkat cell lysate at pH 5.0; lane

5, cytochrome c treated with 10 lg/ml trypsin; and lane 6, 1lg/ml

trypsin. (A) NO2Tyr derivatives of the 23 kDa Mn-SOD and (B) a

longer exposure of nitrated proteins in the cytochrome c region

(13 kDa) of (A). Peroxidase activity corresponding to each condition is

expressed at the bottom, relative to native cytochrome c.

L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107 105

might increase both peroxidase and nitrating activities

of cytochrome c, acidified Jurkat cell lysates were used

as a source of lysosomal protease activity. An increase in

cytochrome c peroxidase activity, Mn-SOD nitration,and cytochrome c self-nitration was observed upon cy-

tochrome c exposure to Jurkat cell lysates at pH 5.0

(Fig. 7B, lane 2 versus lane 4). Less nitration of both

cytochrome c and Mn-SOD was observed when cyto-

chrome c was treated with neutral Jurkat cell lysates,

which also correlated with diminished peroxidase ac-

tivity (Figs. 7A and B, lane 3), possibly also reflecting

H2O2 consumption by other pathways (e.g., cell lysateglutathione peroxidase and catalase). No increase in

nitration (or peroxidase activity) was observed when

cytochrome c was incubated in 100mM phosphate

buffer, pH 5.5, without cell lysates (not shown). As a

positive control, 62.5 lM cytochrome c was incubated

with trypsin for 2 h at 37 �C, also resulting in both

greater peroxidase activity and protein nitration yields

(Figs. 7A and B, lanes 5 and 6).

Discussion

Cytochrome c readily catalyzes NO�2 and H2O2-de-

pendent self- and proximal target molecules nitration,

including protein tyrosines (Figs. 3–5) and low mole-

cular weight phenolics (Fig. 6). Although tyrosinenitration is frequently considered a consequence of

ONOO� production and reaction during tissue inflam-

matory or oxidative injury [9], other mechanisms can

account for this �NO-dependent tyrosine modification in

vivo. In particular, phagocytic cell peroxidases promote

nitration of free and protein-associated tyrosines viaH2O2-dependent oxidation of NO�

2 [14,15,45]. The

present data reveal that a wide spectrum of other per-

oxidase-like hemoproteins, present in diverse cell types,

are capable of catalyzing tyrosine nitration.

The catalytic heme of cytochrome c must bear iron in

a highly oxidized state, as for the compound I of per-

oxidases where a ferryl ion is bound to a porphyrin

cation radical [29]. Formation of �Tyr from the reactionof H2O2 with cytochrome c3þ has been shown by spin

trapping EPR [46]. Then, a rapid electron transfer from

the porphyrin to a vicinal Tyr (i.e., Tyr48 or Tyr67) is

feasible. Indeed, Lawrence et al. [47] using competition

studies involving spin traps have shown that the oxo-

ferryl heme component is the active oxidant species that

would be rapidly quenched by electron transfer to the

protein moiety. For peroxidases such as MPO or EPO,NO�

2 is a substrate for compound I oxidation (rate

constant for MPO¼ 4� 107 M�1 s�1, [17]). Nitrite oxi-

dation occurs during the peroxidatic cycle of MPO in

two separate one-electron steps and yields �NO2 [16,17].

For cytochrome c3þ, the �Tyr formed by the rapid

quenching of the ferryl intermediates can be nitrated by�NO2, oxidize small phenolic substrates and tyrosines in

proteins (this work and [48]), and may be capable ofoxidizing NO�

2 to �NO2 [14]. The presence of cyto-

chrome cmultimers and high molecular weight BSA and

Mn-SOD aggregates, separated by PAGE under reduc-

ing conditions, reveals cytochrome c-induced protein

cross-linking via dityrosine and possibly Schiff�s base

crosslinks (Figs. 1,3–5). This and the separate observa-

tion of peroxidase-induced biHPA formation (Fig. 6)

strongly support the cytochrome c-dependent interme-diate formation of �Tyr and �NO2 radicals. While we

have not directly measured the yield of �NO2 it is ex-

pected to increase when NO�2 concentrations are raised,

leading to higher NO2Tyr formation and lower dityro-

sine cross-linking. The heme dependence of this reaction

is confirmed by inhibition of protein and HPA nitration

by KCN, which displaces the methionine 80 axial heme

ligand [49] and the inhibition of the heme bleachingobserved in the presence of NO�

2 (Fig. 2).

While cyanide abolished MPx 11- and cytochrome

c-dependent BSA nitration, it did not inhibit self-

cytochrome c nitration and polymerization (Fig. 3).

Although we do not have a precise explanation for this

result, it can be speculated that cyanide efficiently blocks

the oxo heme-dependent oxidation of BSA tyrosines,

while at the same time forms short-lived cyanide-derivedradicals, which may locally react with cytochrome c

tyrosines to form �Tyr radicals. These will then combine

with �NO2 to yield NO2Tyr. In fact, the existence of

nitrated cytochrome c in the presence of cyanide clearly

indicates that NO�2 (unlike BSA) was able to compete

with cyanide for binding and reaction at the oxo-

heme site. On the other hand, �NO2 alone will be very

106 L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107

inefficient in promoting tyrosine nitration in BSA, be-cause the process is largely favored with a first oxidation

step to �Tyr, which in this case would be blocked by

cyanide. The formation of cyanide-derived radicals after

reactions with strong oxidants such as hydroxyl radical

has been reported [50,51]. Although the chemical

properties of cyanide-derived radicals remain undefined,

our results suggest that they could promote oxidation

reactions close to their formation sites.The addition of 25mM HCO�

3 to reaction systems (in

equilibrium with 1.3mM CO2) did not lead to greater

nitration yields in cytochrome c-dependent reactions

and also, methionine did not inhibit nitration, ruling out

a role of ONOO� as intermediate in H2O2/NO�2 /cyto-

chrome c-dependent nitration reactions.

Cytochrome c exists in high concentrations in mito-

chondria (>400 lM, [30]). The present in vitro obser-vation of the actions, 200 lM cytochrome c in the

presence of nM/min fluxes of H2O2 and lM NO�2 con-

centrations likely reflects responses expected in vivo,

wherein 10–50 lM levels of NO�2 will accumulate when

high rates of tissue �NO generation, as well as H2O2

production, occur during inflammatory responses. The

significant differences in nitration yields of mitoplasts

depleted and repleted with cytochrome c also support asignificant role for cytochrome c-catalyzed mitochon-

drial protein nitration reactions.

While extensive Mn-SOD nitration was observed

when purified Mn-SOD was incubated with cytochrome

c/H2O2/NO�2 , SOD catalytic activity was not affected

(Fig. 4). An important insight arises from this observa-

tion, since now NO2Tyr formation can be used not only

as a dosimeter of reactive nitrogen species formation,but also to identify the chemical nature of secondary�NO-derived reactive species (i.e., ONOO� versus NO�

2 /

H2O2/peroxidase-derived nitrating species). Human

Mn-SOD contains nine tyrosine residues, with the ex-

clusive nitration of Tyr34, located only a few angstroms

from the active site, responsible for Mn-SOD inactiva-

tion by ONOO� [27,28]. Both the attraction of ONOO�

to the active site, favored by adjacent basic amino acidresidues, as well as the reaction of ONOO� with the

active site Mn, will facilitate the formation of nitrating

species in close proximity to Tyr34 [28,44]. Therefore,

though the Mn-SOD tyrosines modified by cytochrome

c/H2O2/NO�2 were not determined, Tyr34 was excluded

and more solvent-exposed tyrosines (i.e., Tyr9, 11, and

193) became more prominent candidates for nitration by

this peroxidase-catalyzed mechanism. Thus, the con-comitant presence of nitrated and catalytically inacti-

vated Mn-SOD found in rejected human renal allografts

support the formation and mitochondrial reactions of

ONOO� [25].

Protein nitration yields induced by MPx-11 were

greater than those observed with equimolar concentra-

tions of cytochrome c as a catalyst. On the other hand,

NO2HPA formation yields were similar for both cyto-chrome c and MPx-11, emphasizing the fact that nitra-

tion yields of heme protein catalysts will be influenced

by phenolic accessibility to the heme iron and the limited

diffusion distance of the highly reactive �NO2.

The presence of cytochrome c is frequently observed

in the cytoplasmic compartment of various cell types

undergoing apoptosis [34]. Little is known about the fate

and molecular actions of this protein after binding tocytoplasmic APAF-1, but during oxidative stress-in-

duced apoptosis, degradation of cytochrome c was ob-

served during the execution phase, with caspase and

lysosomal protease activities as candidates for mediating

this cytochrome c proteolysis [52–55]. Following partial

proteolysis, cytochrome c peroxidase activity is ampli-

fied, thus its protein nitration capabilities will increase as

well (Fig. 7). Several lines of evidence support the role ofcytochrome c-mediated protein oxidation. For instance,

cytochrome c induced a-synuclein aggregation in the

presence of H2O2 and the co-localization of both cyto-

chrome c and a-synuclein has been shown in Lewy

bodies from Parkinson�s disease patients [56]. Further-

more, protein oxidation of cytochrome c by ONOO�

[36] or by reactive halogen species [57] enhances its

peroxidase activity. Since nitrated proteins are ahallmark of several cell lines and primary cell cultures

undergoing apoptosis [58–60], we postulate that cyto-

chrome c contributes to this protein modification.

Recently it has been shown about the coexistence of

translocated cytochrome c and nitrated proteins in

neurons after oxygen and glucose deprivation [61].

During various pathophysiological situations, the

increased formation of O��2 , H2O2, and

�NO occurs inboth mitochondrial and extramitochondrial compart-

ments. Cytochrome c is shown herein to serve both as a

target and an effector molecule for secondary oxidative

and nitration reactions, both in mitochondria and the

cytosol. The occurrence of NO2Tyr in cells lacking

phagocytic peroxidases will not only reflect ONOO�

production, but also hemoprotein-mediated reactions,

with the amino acid specificity and spatial distributionof nitration reactions providing insight into the source

of oxidizing and nitrating species. The redundancy of

ONOO� [59,62,63] and peroxidases such as MPO, EPO

[45,64], myoglobin [65], and cytochrome c as mediators

of nitration reactions supports the significance of this�NO-dependent post-translational modification in cell

injury and signaling.

Acknowledgments

This work was supported by grants from the National

Institutes of Health (B.A.F.), Fogarty-NIH (B.A.F. and

R.R.) and The Howard Hughes Medical Institute

(R.R.).

L. Castro et al. / Archives of Biochemistry and Biophysics 421 (2004) 99–107 107

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