Clinical Biochemistry 3

Review

Protein S-nitrosation: Biochemistry and characterization of protein

thiol–NO interactions as cellular signals

Shane Miersch, Bulent Mutus*

Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada N9B 3P4

Received 20 January 2005; received in revised form 24 May 2005; accepted 24 May 2005

Available online 11 July 2005

Abstract

The interaction of nitric oxide with thiols is complex and still an active area of research. Herein, we provide an overview of the ways in

which nitric oxide can be biologically transformed into species capable of adding anSNO moiety to protein sulfhydryls, emphasizing how

protein S-nitrosation differs from nitrosation of low molecular weight thiols. Protein S-nitrosation is being revealed as a post-translational

means of chemically modifying and functionally altering proteins. Changes in protein function, which persist on a physiologically relevant

time scale, effectively transmit biological signals and thus provide a framework for elucidating signaling networks. A description of recently

developed methodology facilitating inquiry into this area is provided, along with a sketch of various proteins reported to be targets for

nitrosation and the functional consequences therein. Protein denitrosation appears to be an active and perhaps enzymatically catalyzed

process. Here, we summarize the evidence that suggests this and proffer a precis of proteins possessing denitrosation activity.

D 2005 The Canadian Society of Clinical Chemists. All rights reserved.

Keywords: Protein S-nitrosation; Denitrosation; Nitric oxide; Redox congeners; Post-translational modification; Biotin switch method; Reactive nitrogen

species; Signal transduction

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

Chemistry of nitric oxide, oxygen and S-nitrosothiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

Reaction ofSNO with oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

Reaction ofSNO with superoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779

Transnitrosation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

S-nitrosation reactions of NO with metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

Formation of nitroxyl anion and reaction with thiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

Protein S-nitrosation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

Enzyme-catalyzed S-nitrosation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

Non-enzymatic S-nitrosation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

Protein thiol microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

Proximity of hydrophobic microenvironments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

Metal ion availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

Relative abundance of oxygen and activated oxygen species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

Activity and proximity of nitric oxide generating systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

Identification and characterization of S-nitrosated proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

The biotin switch assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved.

doi:10.1016/j.clinbiochem.2005.05.014

* Corresponding author.

E-mail address: mutusb@uwindsor.ca (B. Mutus).

8 (2005) 777 – 791

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791778

Reported S-nitrosatable proteins and functional significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Thioredoxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Apoptotic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784

Small GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784

Transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

Nuclear factor nB (NFnB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

AP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

Matrix metalloproteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

Viral proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

Denitrosation of low molecular weight thiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

Metal ion-mediated decomposition of nitrosothiols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

Superoxide-mediated decomposition of nitrosothiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

Enzyme-mediated decomposition of nitrosothiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

Denitrosation of S-nitrosated protein thiol residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

Introduction

The intense investigation of the biochemistry of nitric

oxide (SNO) over the last 20 years has yielded a wealth of

both physiological and pathophysiological functions for this

small free radical species. It has been implicated in diverse

roles, including respiration, nerve transmission, apoptosis,

host defense, DNA replication, transcription, modulation of

vascular tone and hemostasis. These functions can be

attributed to the transient interaction of the gaseous diatom

or its redox congeners with transition metal centers, protein

and non-protein sulfhydryls, tyrosine and tryptophan resi-

dues, reactive oxygen species or perhaps to the formation of

lipid-NO or O- and N-nitrosated adducts. However, given

the dynamic nature of nitric oxide and the labile nature of

nitrosated and nitrosylated species, characterization of the

NO-based signals transduced transiently within the cell

remains incomplete. Literature to date focuses largely on

NO signals via the interaction ofSNO and reactive nitrogen

species (RNS) with protein-bound metal centers, protein

thiols or reactive oxygen species. The paradigm of protein

thiol S-nitrosation is generally thought to be in relative

infancy with only a handful of reviews published [8,20,

24,54,66]. Inquiry into this area has only recently benefited

from technical advances in detection and characterization,

based largely upon variations in methodology referred to as

the biotin switch method [38]. This methodology facilitates

study of the process of reversible nitrosation as a post-

translational means of transmitting signals within the cell,

oft likened to phosphorylation. In contrast, regulatory

mechanisms that govern S-nitrosation and denitrosation of

proteins are to date largely non-enzymatic, thus distinguish-

ing the two forms of signal transduction. Several inves-

tigators have made inroads to delineating the functional

significance of NO-based signals transduced through protein

nitrosation [29,41,39,60]. However, suggestion that the

nitrosoproteome may be comprised of greater than 100

proteins [21] indicates that a thorough characterization of

the network of signals transmitted by activeSNO species

remains to be delineated.

This review will include relevant nitric oxide and thiol

chemistries, the role of cellular environment in regulating the

various paths of reaction to yield the resultant S-nitrosated

(SNO) species and proteomic methodology for isolating and

identifying S-nitrosated proteins. It will conclude with an

overview of SNO proteins detected thus far and a discussion

of protein thiol nitrosation as a signaling framework.

Chemistry of nitric oxide, oxygen and S-nitrosothiols

S-nitrosothiols represent a pool of nitric oxide donors

that effectively stabilize the relatively short-lived free

radical, extending the lifetime of the active NO species.

Any protein that bears a free thiol moiety can, in principle,

be S-nitrosated. However, as has been demonstrated,

endogenous nitrosation of protein thiols appears more often

selective. Formation and degradation of S-nitrosothiols is a

dynamic process that is influenced largely by the prevailing

redox environment, oxygen and metal ion availability and

thiol reactivity.

Considering the limited direct reaction betweenSNO and

thiols [104], nitrosothiol formation is generally preceded by

reaction with other species. Thus, an understanding of

protein S-nitrosation is predicated upon knowledge of the

chemistry of nitric oxide and its redox congeners with

oxygen, metal ions and reactive oxygen species.

Reaction ofSNO with oxygen

An interesting aspect of nitric oxide chemistry is the rela-

tive unreactive nature ofSNO to most biological molecules,

excluding other free radicals and metal centers. It has been

demonstrated thatSNO will not nitrosate low molecular

weight thiols, such as cysteine and glutathione, in the absence

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 779

of oxygen and must be ‘‘activated’’ in order to participate in

S-nitrosation reactions; thus, a primary route ofSNO ‘‘acti-

vation’’ is via reaction with diradical oxygen. The reaction ofSNO with oxygen in an aqueous environment is as follows:

4SNOþ O2 þ 2H2O Y 4NO�

2 þ 4Hþ ð1Þ

k ¼ 2� 106 M�2s�1 at 25-C� �

[58]

and the kinetic rate of disappearance ofSNO has been deter-

mined to be second order with respect toSNO concentration.

This assertion bears several implications; firstly, that this

reaction will predominate at higher fluxes ofSNO; secondly,

that at low oxygen concentrations, oxygen will act to limit the

reaction; and thirdly thatSNO and O2, both being hydro-

phobic gases, will concentrate within the hydrophobic inte-

rior of the biological compartments, thus facilitating reaction.

Indeed, it has been suggested by Liu et al. that the auto-

oxidation of nitric oxide is confined (>90%) to hydrophobic

compartments [58,105]. This overall reaction is comprised of

several intermediate reactions, represented as follows:

2SNOþ O2 Y 2NO2S ð2Þ

NO2Sþ NO2S Y N2O4 ð3Þ

k ¼ 4:5� 108 M�1s�1� �

[23].

N2O4 is capable of S-nitrosation via the reaction:

N2O4 þ RSH Y RSNOþ NO�3 þ Hþ ð4Þ

[23]. However, in aqueous systems, water will compete with

nitrosation to effect hydrolysis, seen below:

N2O4 þ H2O Y NO�2 þ NO�

3 þ 2Hþ ð5Þ

k ¼ 1� 103 s�1� �

[23].

Nitrogen dioxide radical also participates in further reaction

withSNO forming nitrous anhydride as follows:

SNOþ NO2S Y N2O3 ð6Þk ¼ 1:1� 109 M�1s�1� �

[23].

Nitrous anhydride (N2O3) is a potent nitrosating agent

capable of transferring nitrosonium equivalents (NO+) to

thiols, thus generating both low molecular S-nitrosothiols

and S-nitrosoproteins via the general reaction scheme:

N2O3 þ RSH Y RSNO þ NO�2 þ Hþ ð7Þ

k ¼ 6:6� 107 M�s�1� �

[44].

Although the rate constant provided was determined for

glutathione, it is expected that protein microenvironments,

whether hydrophobic/hydrophilic or acidic/basic, would

greatly influence the rate of formation of nitrosated pro-

tein thiols. Nitrous anhydride, like dinitrogen tetroxide, is

subject to hydrolysis reactions in aqueous environments,

as shown in the following reaction:

N2O3 þ H2O Y 2NO�2 þ 2Hþ ð8Þ

k ¼ 1� 103 s�1� �

[23].

N2O3 is also formed by the reaction of protonated nitrite in

acidic microenvironments (such as peroxisomes and lyso-

somes). The reaction is essentially a dehydration reaction in

which two molecules of nitrous acid lose water and generate

the nitrosating species:

Hþ þ NO�2 Y HNO2 ð9Þ

[23]

2HNO2 Y N2O3 þ H2O ð10Þ

[23]

In light of the high concentrations ofSNO required for

autooxidation reactions, Gow et al. have provided evidence

suggesting an alternate mechanism that would operate at

physiologicalSNO concentrations [26]. This mechanism,

shown below, proceeds via a radical intermediate which

donates an electron to an electron acceptor, thus producing

nitrosothiol [26].

RSHþ SNO Y RSNS� OH ð11Þ

RSNS� OHþ O2 Y RSN ¼ OþO2

S� ð12Þ

One implication of this mechanism is that the reaction

will proceed under anaerobic conditions, but only in the

presence of an electron acceptor such as NAD+.

Reaction ofSNO with superoxide

As indicated,SNO autooxidation can proceed via a

reaction with higher order dependence on nitric oxide. Thus,

this reaction would proceed primarily under circumstances

of increased localized concentration of nitric oxide. Given

the stoichiometric limitations of this reaction, other reaction

pathways for the consumption/activation of nitric oxide may

play a significant role in its chemical transformation.

Importantly,SNO reacts with superoxide at diffusion-limited

rates, forming peroxynitrite when both reactants are

supplied at equimolar levels.

SNOþ O2

S� Y OONO� ð13Þ

k ¼ 6:7� 109 M�1s�1� �

[6].

The physiological relevance of this reaction is supported by

evidence thatSNO synthases can, under conditions of

arginine scarcity, produce superoxide as well as nitric oxide

[43]. Once protonated, peroxynitrite forms peroxynitrous

acid which can undergo homolytic decomposition, forming

hydroxyl radical and nitrogen dioxide.

OONO� þ Hþ Y OONOH Y OHSþ NO2S ð14Þ

[82]. Both generated hydroxyl radical, as well as nitrogen

dioxide, and perhaps peroxynitrite itself [81] are capable of

abstracting a proton from available thiols yielding the thiyl

radical (RSS). Thiyl radical is then subject to quenching

reactions with nitric oxide yielding the S-nitrosated thiol

analog. Despite that peroxynitrite is generally considered a

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791780

potent and deleterious oxidant, direct interaction of perox-

ynitrite with thiols yielding S-nitrosothiols has been demon-

strated [101]. This reaction appears to involve nucleophilic

substitution at the thiol with elimination of HOO�,

evidenced by the formation and detection of H2O2. S-

nitrosation was observed to take place in the presence of

physiological levels of carbonate/carbon dioxide and

although yields achieved were only as high as 5%, given

the high intracellular protein and non-protein thiol concen-

tration, contribution of this reaction to intracellular S-

nitrosothiol levels may be significant. Evidence for a role

for peroxynitrite in protein thiol nitrosation has been

obtained through the treatment of sarcoplasmic reticulum

vesicles with varying concentrations of OONO� [102],

resulting in nitrosation of specific cysteine residues that may

be responsible for functional modulation.

Transnitrosation

The nitric oxide moiety of S-nitrosothiols exchanges

readily with other thiols, preferentially over nitrogen or

carbon, via nucleophilic attack of the S–NO nitrogen by

thiolate anion:

R1SHþ R2SNO Y R1SNOþ R2SH ð15Þ

Thus, reactivity of thiols correlates well with increased

acidity of the thiol group and would thus have significant

effect on the transnitrosation of protein thiols from small

molecular weight nitrosothiols, given the influence of

protein microenvironment. Spectral studies conducted to

investigate the kinetics of transnitrosation between low

molecular weight thiols, such as glutathione and cysteine,

andSNO donors, such as S-nitrosoacetylpenicillamine

(SNAP), revealed that rates of transnitrosation were faster

than decomposition yieldingSNO at physiological pH [2].

This suggests first that heterolytic mechanisms of decom-

position play a physiological role in consumption of S-

nitrosothiols, and second that the ease with which transfer of

the NO-moiety occurs increases the likelihood that thiol-

based storage pools of nitric oxide are involved in regulation

of protein function by S-nitrosation reactions. Thiols are

also known to undergo reaction with nitrosothiols in

manners other than transnitrosation and are discussed below.

These above observations indicate that if S-nitrosated

proteins are to persist in this form on a time scale that can

influence cellular physiology, the protein must likely be

sequestered away from the cytoplasmic compartment due to

the prevalence of thiol reductants that would promote its

rapid decomposition.

S-nitrosation reactions of NO with metals

The coordination ofSNO by metals forming metal–

nitrosyl complexes has been well studied. As a metal ligand,

SNO is capable of assuming either electrophilic (by donating

an electron to the metal) or nucleophilic (by accepting an

electron from the metal) character. Hemoproteins (guanylate

cyclase, hemoglobin, cytochrome P450) constitute some of

the best studied systems of metal–nitrosyl interactions.

Iron-based metal–nitrosyl complexes are capable of nitro-

sating thiols through a redox reaction in which a ferric–

nitrosyl is reduced to its ferrous state with concomitant

transfer of nitrosonium to a free thiol:

Fe3þ haemð Þ þ SNO Y Fe2þ � NOþð Þhaemþ RSH

Y RSNOþ Fe2þhaemþ Hþ ð16Þ

[103]. Stubauer has noted that free cupric ion efficiently

catalyzes S-nitrosation of free thiols in both bovine serum

albumin and human hemoglobin, but it is ineffective in

catalyzing transfer of theSNO moiety to the low molecular

weight thiol glutathione as a result of the rapid disulfide

bond formation catalyzed by Cu2+ [94]. Regardless, the

limited availability of unbound copper [83] would likely

limit applicability of this reaction intracellularly.

Formation of nitroxyl anion and reaction with thiols

Nitroxyl anion (NO� or HNO at physiological pH on the

basis of its pKa) is the reduced congener of nitric oxide, and

although its chemical existence has been recognized for

over a century, its chemistry is complicated and its

biochemistry is still an area of active inquiry. Recent studies

reveal thatSNO and HNO can have distinct pharmacolog-

ical actions [70]. It has been shown thatSNO and HNO

undergo interconversion in vitro; however, the direct one-

electron reduction of nitric oxide in vivo is believed to be

highly unfavorable [4]. Despite the unlikelihood of for-

mation in this manner, HNO has been shown to be formed

upon reaction of thiols with nitrosothiols. Nucleophilic

attack of the RSNO thiol by reduced thiol yields the

oxidized disulfide and nitroxyl anion:

RSNO þ RSH Y RSSR þ HNO ð17Þ

[106]. Alternatively, Hobbs et al. found that the addition of

superoxide dismutase (SOD) to nitric oxide synthase resulted

in an enhanced generation of nitric oxide (as analyzed by

chemiluminescence) that could not be accounted for by

additional generation of citrulline [34]. The authors sug-

gested that additionalSNO was being formed via SOD-

catalyzed oxidation of nitroxyl produced enzymatically [34].

Indeed, one electron oxidation of HNO to nitric oxide is

catalyzed not only by superoxide dismutase (SOD), but also

by a variety of other ubiquitous biological oxidants [22,73].

Literature reports of the direct reaction of HNO with

thiols indicate the formation of sulfonamide adducts [106],

yet reaction of nitroxyl anion with electropositive protein

thiols also appears to plays a role in nitrosation of the

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 781

NMDA receptor at the Cys399 residue of the NR2A subunit

[45,56].

Protein S-nitrosation

In order to characterize the relative contribution of

nitrosation versus nitrosylation reactions on a global scale,

Feelisch measured S-nitrosation, N-nitrosation and metal–

nitrosyl formation in rat plasma, red blood cells, heart, lung,

aorta, liver, kidney and brain tissues [9]. Their study

demonstrated the prevalence of S-nitrosation products in

all tissues, in contrast to the limited localization of metal–

nitrosyl products to the brain, heart, liver and kidney [9]. In

those tissues where both were found, it is important to note

that nitrosation products were found at levels comparable to

nitrosylation products, and that the majority of nitrosation

products were associated with the protein fraction.

The results of their study reveal that protein S-nitrosation

is a ubiquitous occurrence that rivals the competing process

of metal nitrosylation, despite appearances of being kineti-

cally disfavored. It further underscores the possibility that

protein S-nitrosation could be a widely used mode of

transmitting cellular signals, analogous to protein phosphor-

ylation, especially when one considers the dynamic nature

of nitrosation and denitrosation.

Enzyme-catalyzed S-nitrosation

Despite suggestions of a role for particular S-nitrosatable

protein cysteine residues in transfer of anSNO moiety to

other proteins [31], experimental evidence of substrate-

specific protein nitrosation or transnitrosation has not, to our

knowledge, been forthcoming. Several proteins however

have been reported to be capable of catalyzing intra-

molecular and intermolecular S-nitrosation reactions. For

example, ceruloplasmin, a multi-copper containing oxidase

protein found abundantly in plasma, has been shown to

efficiently catalyze the S-nitrosation of low molecular

weight thiol compounds such as glutathione and N-acetyl

cysteine via a one-electron oxidation of nitric oxide [37].

Whether this enzyme plays any role in S-nitrosation of

protein thiols, however, has yet to be determined.

Nudler has shown that bovine serum albumin is capable

of catalyzing the S-nitrosation of low molecular weight

thiols through a mechanism that involves the selective

partitioning of the nitrosating species, N2O3, into the

hydrophobic pockets of the protein [84]. Interestingly,

micellar catalysis of thiol nitrosation has also been

demonstrated in protein disulfide isomerase [91]. Although

in vivo catalysis of S-nitrosation of protein thiols through

nitrosating equivalents stored within hydrophobic micro-

environments of other proteins is conceivable, evidence is

not yet forthcoming. This may, however, represent a general

mechanism by which nitrosation equivalents stored within

hydrophobic protein compartments are transferred to low

molecular weight thiols and can then react with protein

thiols through transnitrosation.

As indicated, iron–nitrosyl compounds are capable of

transferring nitrosonium equivalents to available thiols: a

reaction important in autocatalytic S-nitrosation of Cysh93in human hemoglobin [41,93]. Although this has been

controversial, it appears that it indeed plays a role in the

redox-catalyzed nitrosation of this particular residue. Recent

experiments have attempted to account for the variation

responsible for the controversy and show that S-nitrosation

of HbCysh93 may depend on the rates at which nitric oxide

is added, the volumes of solutions used and buffer

concentrations and concluded that nitrosation occurs via

N2O3,SNO2 and metal-mediated pathways [33].

Given the relative paucity of enzyme-mediated S-nitro-

sation reactions in the literature to date, protein–thiol

nitrosation reactions may be governed largely by non-

enzymatic mechanisms.

Non-enzymatic S-nitrosation

S-nitrosation of protein sulfhydryls can differ signifi-

cantly from the nitrosation of low-molecular weight thiols

and are influenced by a variety of factors outlined below.

Protein thiol microenvironment

It has been noted that proteins with significant free thiol

content are not nitrosated to an extent that would be

expected, if all free thiols were equally nitrosatable. Indeed,

several studies that have isolated S-nitrosatable proteins

found few, if any, cysteine-rich proteins [40]. It is, however,

possible that proteins bearing closely spaced free thiols may

be nitrosatable, but that nitrosation of a single free thiol

would then be susceptible to denitrosation by other thiols in

close proximity. We have observed, for instance, that

stoichiometrically nitrosated protein disulfide isomerase is

stable over a period of 2 h [91], yet substoichiometric

nitrosation of available thiols results in rapid decomposition

of available S–NO bonds and concomitant disulfide bond

formation in an autocatalytic manner (unpublished obser-

vations). Nonetheless, the seemingly selective nature of

protein S-nitrosation has led to the suggestion of a

consensus motif for cysteine reactivity of proteins by the

Stamler group [92]. It is well known that protein residues in

close proximity to cysteine can influence thiol reactivity by

general acid-base catalysis. Inspection of a body of known

S-nitrosatable proteins reveals the repeated presence of an

acidic amino acid immediately following, as well as an

acidic or basic residue immediately preceding reactive

cysteines in a significant number of proteins [92]. Further

analysis suggested that a polar amino acid in the Cys-2

position may also have some predictive ability and thus a 3-

residue degenerate consensus motif for S-nitrosation as

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791782

(G,S,T,C,Y,N,Q)(K,R,H,D,E)(C)(D,E), analogous to that of

glycosylation or phosphorylation was constructed. Though

it has been asserted that the most important component of

the consensus sequence is the Cys (Asp/Glu) pair, the role of

the three dimensional microenvironment of a reactive thiol

would similarly provide important predictive clues to

enhanced nitrosative susceptibility [3].

Proximity of hydrophobic microenvironments

In addition to changes in thiol behavior, both protein and

cellular microenvironments would also influence the

autooxidation and nitrosative ability of nitric oxide.

Importantly, the enhanced solubility of bothSNO and O2

in hydrophobic biological compartments has been shown to

facilitate autooxidation by helping to overcome the third

order requirement ofSNO in formation of the potent

nitrosating species nitrous anhydride [58]. Although it is

likely that thiols localized to transmembrane domains or low

dielectric regions of a protein would be subject to nitro-

sation by agents preferentially formed in lipid environments,

to our knowledge there has been no specific reports of this

in the context of signal transduction. However, as testament

to the ability of protein hydrophobic compartments to store

NO equivalents, it has been shown that bovine serum

albumin is capable of micellar catalysis of low molecular

weight S-nitrosothiol formation [84]. Indeed, protein disul-

fide isomerase has been found similarly capable of storing

nitrosating equivalents and catalyzing nitrosation, independ-

ent of protein thiols [91]. In light of these observations, this

may be a generalized form ofSNO storage and transfer of

NO+ to protein thiols via nitrosated low molecular weight

thiols.

Metal ion availability

Catalytic formation and decomposition of S-nitrosothiols

has been shown to occur in aqueous buffers via reaction

with copper ions. Experiments have shown rapid reaction ofSNO with protein thiols in both bovine serum albumin and

human hemoglobin in the presence of Cu2+ [94], forming

near stoichiometric amounts of protein S–NO in either

oxygenated or deoxygenated solution. In the course of

nitrosothiol formation, cuprous ion accumulates and causes

the destabilization of S–NO bonds. However, the physio-

logical availability of free copper ions is doubtful [83], thus

limiting the applicability of copper-catalyzed S-nitrosation.

Calcium ions have recently been ascribed responsibility for

the regulating nitrosation and denitrosation of the multi-

functional tissue transglutaminase (tTG) [50]. tTG is highly

expressed in endothelial cells, bears an exposed Cys277 in

the presence of Ca2+ [28] and has been shown to be a target

for S-nitrosylation [69]. Lai et al. showed that nitrosation of

tTG could be increased from 7.2 mmol to 14.5 mmol of

nitrosothiol/mol of tTG by the addition of 8 mM Ca2+ and

that incubation of recombinant enzyme with endothelial

cells plus ionophore resulted in a significant increase in

nitrosation over controls [50].

Although it does not appear as though the authors made

efforts to characterize or control potential copper contam-

ination, this work may represent a general means by which

physiologically available ions can regulate nitrosative status

of a protein.

Relative abundance of oxygen and activated oxygen species

Specificity of protein S-nitrosation reactions is likely

achieved in part by modulating the chemical identity of the

various S-nitrosating species derived fromSNO. The role of

reactive oxygen species in activating nitric oxide towards

thiol nitrosation is suggested in a recent study by Bryan et

al. [9] and also by the third order requirement ofSNO (see

reactions 1 and 6 above) and thus the relatively high levels

ofSNO required to elicit formation of the N2O3 nitrosating

species. The importance of the diffusion-limited reaction

betweenSNO and superoxide is outlined by Espey et al. in

which the rate of reactionSNO with the

SNO-sensitive

fluorophores diaminonaphthalene (DAN) [48] and diami-

nofluorescein (DAF) [47] was measured against a back-

ground of superoxide generation at physiological (nM)

levels [17]. Product formation was increased as the rate of

superoxide production approached equality with that of

nitric oxide production and dropped off precipitously as

superoxide production exceeded that ofSNO [17]. Further,

recent papers point to the ability of mitochondrial impair-

ment and peroxynitrite scavenging to influence formation

of S-nitrosated proteins [108]. As a rich source of reactive

oxygen species, it is conceivable that the mitochondria may

be a primary locale for production of S-nitrosoproteins

[62].

Activity and proximity of nitric oxide generating systems

Evidence has been presented supporting the view of

protein S-nitrosation as dynamic process closely linked to

the activity of the various NOS isoforms on downstream

protein target S-nitrosation [27]. Gow et al. employed

antiserum raised against S-nitrosated bovine serum albumin

to show a concomitant increase in immunoreactivity in both

bovine pulmonary artery and rat CPA-47 endothelial cells

following stimulation with calcium ionophore A23187 [27].

Increases in immunoreactivity could be largely attenuated

by both treatment with mercury and pre-treatment with NG-

monomethyl-l-arginine acetate (l-NMMA) [27]. Immunor-

eactivity was similarly inhibited in stimulated murine aortic

slices, PC-12 and RAW 254.7 cells pre-treated with l-

NMMA [27]. Several groups have suggested that specificity

may be in part determined by colocalization or close

proximity of protein S-nitrosation targets with nitric oxide

synthase isoforms as a means of facilitating reaction

[92,40,113]. The contemporaneous observation of a mito-

chondrial isoform of NOS (mtNOS) [32] and the prevalence

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 783

of S-nitrosated proteins in the mitochondria and peri-

mitochondrial region [108] may support these assertions.

Identification and characterization of S-nitrosated

proteins

S-nitrosation of proteins can be thought of as analogous

to protein phosphorylation, as chemical modification with

either NO or PO43� is a transient occurrence that typically

modifies only a subpopulation of a particular protein.

Therefore, methodology must address the analytic chal-

lenges of lowly abundant modified proteins in in vivo

systems to achieve sufficiently sensitive analyses.

The study of the phosphoproteome has been facilitated

by use of phosphomotif-directed antibodies. Similar anti-

bodies, though incompletely characterized and potentially

problematic due to epitope instability, exist for S-nitrosated

cysteine and have been used in several studies of protein S-

nitrosation [7,27,31,59]. Methodology has recently been

developed which allows for selective enrichment and

identification of S-nitrosatable proteins.

The biotin switch assay

The biotin switch assay is of utility to investigators in the

study of protein S-nitrosation as a post-translational means

of cellular communication. This methodology, pioneered by

Jaffrey et al. [38], has been used to successfully identify

several endogenously nitrosated proteins, including the

NMDA receptor [39], caspase-3 [60], catalase and dihy-

drolipoamide dehydrogenase [21]. Using the biotin switch

assay, investigators have isolated and identified proteins

endogenously S-nitrosated from cells under conditions of

basalSNO production [60], via stimulation of cellular NOS

[46] and via comparison to nNOS knockout mice [39]. This

methodology exploits the loss of reactivity of the thiol

moiety with various thiol-reactive reagents upon S-nitro-

sation. The neutral thiol-reactive agent methyl methanethio-

sulfonate (MMTS) has been used most widely in effectively

blocking free sulfhydryls while leaving S-nitrosated thiols

and disulfide bonds untouched [38,21,49]. Protein mixtures

are generally treated with denaturing agents such as sodium

dodecyl sulfate, thus ensuring access of MMTS to all thiols,

including those buried within the protein core. Protection

from light, which can cause decomposition of the S–NO

bond, is an imperative to ensure sensitivity of the method-

ology. Following thiol methylation, nitric oxide is cleaved

from available �SNO moieties through photolysis or

chemical reduction with ascorbate. Removal of unreacted

MMTS can be easily accomplished by precipitation with

organic solvents or via use of spin concentrators and is

necessary to avoid reaction of MMTS with the biotin label.

Newly available, reduced thiols are then reacted with a

thiol-specific biotinylating agent such as biotin–HPDP (N-

(6-(biotinamido)hexyl]-3V-(2V-pyridyldithio)propionamide)),

thus facilitating isolation of biotin-modified proteins on a

streptavidin-stationary phase. Streptavidin-bound proteins

from cellular sources are comprised of both SNO-modified

as well as endogenously biotinylated proteins, thus warrant-

ing a caveat for investigators determining nitrosatability of

protein sulfhydryls in vivo. Affinity-labeled proteins are

then separated and identified by mass spectrometric

analysis.

Emerging variations on this method demonstrate its

versatility in the study of nitrosation signaling. Two groups

report the application of modified methods that employ

similar blocking/reduction/labeling strategies in intact cells

and tissues and report the localization to both the

mitochondrial [108] and nuclear regions [10]. Jaffrey et al.

has also amended the biotin-switch method by substituting

an S35 label in place of biotin [40] to facilitate mapping of

nitrosated protein thiols. Substitution of [35S]-2-amino-3-(2-

pyridyldithio)-propionate (APDP) for the biotin–HPDP

label following reduction of protein SNO moieties enables

investigators to visualize hot labeled protein fragments on

two-dimensional gels of tryptic digests, indicative of protein

thiol nitrosation.

Reported S-nitrosatable proteins and functional

significance

Thioredoxin

Thioredoxin (Trx) is a member of a family of redox-

sensitive proteins that bear a conserved vicinal thiol motif

(positions 32 and 35), critical to their role in redox

regulation within their catalytic active sites [80]. Trx also

bears an additional cysteine residue at position 69, which is

essential for maintaining its redox regulatory and anti-

apoptotic functions [31]. Similar to PDI, Trx participates in

electron transfer reactions via the reversible formation of a

disulfide bond between closely spaced sulfhydryls. The

close proximity of these residues, in concert with enhanced

chemical reactivity, provides a redox-sensitive active site

that is influenced by the prevailing oxidative and nitrosative

conditions. Not surprisingly, this ubiquitously expressed

protein [36] has been shown to be nitrosatable [31]. A study

has revealed that overexpression of Trx in endothelial cells

resulted in an overall increase in intracellular SNO content

in protein fractions that was reduced by inhibition with NOS

inhibitors [31]. Similar results were obtained using anti-

sense oligonucleotides directed against Trx. Further, trans-

fection of endothelial cells with either wild-type Trx or a

C32/35S Trx mutant resulted in an increase in protein SNO

content whereas the C69S Trx mutant resulted in no change

in SNO protein content compared to cells treated with

vector alone. Western blots for affinity-tagged Trx and

protein S-nitrosation showed colocalization of S-nitrosated

protein with both wild-type and C32/35S mutant Trx and the

absence of S-nitrosated protein with C69S mutant, leading

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791784

the authors to conclude that C69 was the nitrosatable thiol

[31]. Activity of purified Trx was monitored via consump-

tion of NADPH at 340 nm in order to investigate the

influence C69 S-nitrosation by the NO donor papanoate. In

summary, it appears as though S-nitrosation of C69 results

in a substantial increase in Trx activity versus control, and

that this increase is not inducible in the C69S mutant. A

possible explanation for the observed increase in Trx

activity stems from studies on the ability of the thioredoxin

system (thioredoxin and thioredoxin reductase (TR) to

denitrosate S-nitrosoglutathione (GSNO)) [74]. It is con-

ceivable that the increase in the activity of thioredoxin

observed could be attributed to denitrosation of Cys69

catalyzed by the thioredoxin system itself. As an additional

substrate that could be responsible for consumption of

NADPH, this would represent an alternate and interesting

explanation for the observed phenomena; however, SNO

content was not reported following the assay. In order to

determine the effects of endogenous S-nitrosation on Trx

activity, endothelial cells transfected with either wild-type

Trx or the C69S mutant were simultaneously treated with

the NOS inhibitor, l-NMMA. In agreement with in vitro

results, cellular Trx exhibited reduced activity upon

inhibition of NOS in wild-type transfected cells that was

not observed in cells transfected with C69S mutants [31]. In

support of these results, investigators from another group

were able to isolate S-nitrosated thioredoxin from HEK-293

cells treated with 1 mM exogenous GSNO for 8 h [96] and

additionally that S-nitrosation appears to induce dissociation

of apoptosis signal regulating kinase 1 [109].

The thioredoxin superfamily encompasses a broad range

of proteins related by the presence of a redox-active C-X-X-

C tetrapeptide motif and the highly conserved thioredoxin

fold [19]. Notably, the enzyme protein disulfide isomerase, a

member of the thioredoxin superfamily, has also been

shown to be nitrosatable in vitro [111]. Recent mechanistic

investigations have further shown that protein disulfide

isomerase (PDI) is nitrosatable and that nitrosation with a

fivefold excess of theSNO donor, diethylamine NONOate,

resulted in complete loss of PDI-catalyzed insulin reduction

activity [91]. These results suggest that active site thiols are

being targeted and also revealed that PDI–SNO is stable on

the scale of hours [91]. As an abundant cellular protein, PDI

may be endogenously S-nitrosated; however, to date, there

has been no data presented indicating existence of stable

PDI–SNO, in vivo. Alternately, the observation that treat-

ment of HEL cells with increasing concentrations of SNO

(via SNO–Sepharose beads) substantially reduces PDI

folding activity [111] is suggestive.

Apoptotic proteins

Caspases are a family of proteolytic enzymes that

execute the program for cellular death.SNO is believed to

exert a portion of its anti-apoptotic effects through nitro-

sation of the active site cysteine of at least two, and perhaps

more, of the members of the caspase family [16]. Cys163 is

the active site cysteine of caspase-3 found within the p17

subunit of the heterodimer formed following cleavage of the

zymogen. In an attempt to identify the specific site of S-

nitrosation, investigators employed a p17-myc construct

transfected into COS-7 cells. Subsequent treatment of the

cells with high (1 mM) concentrations of Cys-NO or 50 AMsodium nitroprusside resulted in the formation of an S-

nitrosated p17-myc chimera [86], as detected by NO-spin

trap adducts formed following liberation of nitric oxide from

immunoprecipitates. Introduction of a C163A mutation

abolished detectable S-nitrosation of the construct following

treatment of cells with exogenous NO donor.

Endogenous S-nitrosation of the caspase-3 zymogen has

also been demonstrated in human lymphocytes which

express nitric oxide synthase [60]. In this study, immuno-

precipitated caspase-3 displayed significant nitric oxide

release during photolysis chemiluminescence in comparison

to immunoglobulin controls [60]. Notably, this signal could

be reduced to control levels by pre-treatment of the

immunoprecipitates by HgCl2 [60]. Concerns have been

raised about these results, in light of the use of whole

immunoprecipitates, and the possible contribution ofSNO

from other S-nitrosated proteins which co-immunoprecipi-

tate [46]. Nevertheless, further to these observations, it has

been shown that doxorubicin-induced apoptosis in cardio-

myocytes could be attenuated by treatment of cells with

exogenous SNAP. The protective effect of SNAP was

largely lost by addition of the SNO-decomposing agent,

mercuric chloride, and was also shown to reduce accumu-

lation of cleaved caspase-3 [59]. This paradigm of

protection against apoptosis by caspase S-nitrosation may

extend to other members of the caspase family. Further

evidence of the role of nitric oxide in apoptosis stems from

studies of the initiator caspase–procaspase-9 in HT-29

human colon adenocarcinoma cells. Procaspase-9 was found

to be endogenously S-nitrosated following immunoprecipi-

tation and visualization by the biotin switch method [46]. In

support of a role for nitric oxide in stabilization of caspase

zymogens, cells treated with both tumor necrosis factor

alpha (TNF-a) and the NOS inhibitor, l-NMMA, exhibited

enhanced cleavage of procaspase-9 in comparison to TNF-a

alone.

Small GTPases

Mutagenesis studies on the ras oncogene product, p21ras,

have revealed a single site of nitrosation at Cys118 [53].

Nitrosation of this guanine nucleotide binding protein is

associated with an enhancement of the rate of GTP

hydrolysis and increased levels of cellular Ras-GTP. P21ras

is known to play roles in diverse cellular processes including

growth, differentiation and apoptosis [51,104]; thus, it has

been suggested that the observed influence of nitric oxide in

these same processes may be, in part, mediated through

modulation of the activity of the G-protein [52]. Structural

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 785

studies of p21ras have revealed that Cys118 is the most

solvent-exposed free sulfhydryls [104], and the remaining

thiols lie in regions of the protein better shielded from

solvent exposure, thus alluding to the role of accessibility in

protein S-nitrosation. Topological analysis of p21ras reveals

that Cys118 lies within the highly conserved NKXD (X

being cysteine) region which forms part of the GDP/GTP

binding pocket [104]. However, direct interaction between

Cys118 and either bound nucleotide or other critical residues

within the binding pocket is reported not to occur [104]. In

an effort to determine whether structural perturbations occur

upon nitrosation of Cys118 NMR spectra of Ras were

compared to the spectra of its nitrosated analog [104].1H–15N heteronuclear single quantum coherence and 15N-

edited 3D nuclear Overhauser enhancement experiments

revealed minor changes in chemical shifts that were

restricted to several residues in close 3D proximity to

Cys118, but revealed no major changes in secondary or

tertiary structure that would account for changes in guanine

nucleotide exchange (GNE) activity [104]. Interestingly,

stably S-nitrosated Ras showed no differences in GNE

activity; however, if NO donors (CysNO, GSNO) were

added to the reaction mixture simultaneously, the expected

increase in activity was observed. The authors concluded that

the chemical process of S-nitrosation and not the stably S-

nitrosated product was responsible for observed increases in

GNE activity.

Other GTPases have also been identified as targets for

nitrosation, including the regulators of nucleocytoplasmic

transport Ran [10] and Dexras1 [40]. Although it is unknown

whether this is a regulatory mechanism applicable to other

proteins, additional G-proteins have been found to interact

with various isoforms of nitric oxide synthase (NOS) [40],

again raising the question as to whether proximity of NOS to

nitrosation targets is a means of regulation.

Transcription factors

Numerous examples of ways in which S-nitrosation of

transcription factors can modulate transcription events have

been suggested [1,65,75,95]. However, the physiological

significance of these interactions often remains unclear as a

result of cell-specific differences in response, effects which

vary dependent upon the concentration ofSNO, as well as

redox modulation of nitric oxide under prevailing conditions.

Undoubtedly, some of the variability can also be attributed to

the complexity and redundancy of intracellular signaling

networks that culminate in transcription. Much work remains

to be done in order to thoroughly characterize the role of S-

nitrosation in modulation of transcription events. Noted

below are examples that have initiated this area of inquiry.

Nuclear factor jB (NFjB)

NFnB is a redox-sensitive pro-inflammatory heterodi-

meric transcription factor ubiquitously expressed in the

cytoplasm of all cells [90]. Activation of NFnB results in its

translocation into the nucleus where it is believed to target

over 200 genes, including the NOS2 gene [107] as well as a

variety of genes involved in the inflammatory response [63].SNO-induced inhibition of NFnB transcriptional activity has

been reported [68] in human lymphoblastoid T cells [88], in

murine macrophages [13], in human vascular smooth

muscle cells [89] and in rat astroglial cells [77]. In an

elaboration on the mechanism of observed inhibition, de la

Torre et al. have characterized the effects of S-nitrosation of

the p50 subunit of NFnB on its equilibrium DNA binding

constant [14]. Gel-shift assays using the NFnB target

oligonucleotide revealed an approximately fourfold

decrease in affinity for the target sequence following S-

nitrosation versus non-nitrosated controls.

Marshall et al. have further shown that induction of A549

and RAW 264.7 cells with either tumor necrosis factor-a or

lipopolysaccharide followed by treatment with S-nitro-

socysteine resulted in decreased NFnB transcriptional

activity by a luciferase reporter assay [64,67]. This was

corroborated by electrophoretic mobility shift assays in

which nuclear extracts displayed reduced binding of p50

and p65 subunits of NFnB to the NFnB consensus

oligonucleotide following treatment of cells with S-nitro-

socysteine [64,67]. As expected, binding could be restored

upon treatment of nuclear extracts with dithiothreitol [64].

In contrast, inhibition of NOS using N-monomethyl-l-

arginine (NMMA) in A549 cells was associated with

increased binding of extracted NFnB heterodimer to the

consensus sequence, an effect that could not be mimicked

with the D-isomer NMMA [64,67].

Redox sensitivity of transcriptional events mediated by

NFnB has been attributed to a conserved Cys62 of the p50

subunit, prompting investigation of its susceptibility to

nitrosation [68]. As expected, modification of this particular

residue could be elicited by treatment withSNO gas and was

detected as a 29-Da shift in the molecular weight of the

peptide by electrospray mass spectrometry [68]. Although at

least one role for nitrosation of NFnB at its critical cysteine

residue appears to be a feedback mechanism that limits the

expression of NOS, other physiological roles for this

interaction require elaboration.

AP-1

Similar to NFnB, the subunits c-jun and c-fos of the AP-

1 transcription factor heterodimer are reported to each bear a

single conserved cysteine residue (Cys-272 and 154,

respectively) that confers redox sensitivity to transcription

[1]. NO-mediated regulation of AP-1 transcriptional activity

has been demonstrated by the in vitro treatment of nuclear

extracts from both mouse cerebellar granule cells and

NIH3T3 cells with sodium nitroprusside (SNP) [97].

Analysis of DNA binding gel shift assays revealed, similar

to NFnB, that SNP treatment resulted in inhibition of target

oligonucleotide binding [97]. Nikitovic et al. used the AP-1

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791786

heterodimer comprised of truncated polypeptides for both

Jun and Fos containing the leucine zipper, DNA binding

domains and conserved cysteine residues and compared the

DNA binding activity to that of cysteine to serine mutants in

the presence or absence of varying concentrations ofSNO

[75]. Inhibition of DNA binding was observed and varied

withSNO in a concentration-dependent manner [75]. In

contrast, DNA binding by the cysteine to serine mutant

heterodimer bound the target oligonucleotide with no

sensitivity to treatment with nitric oxide, thus leading

investigators to conclude that inhibition of binding was

mediated by nitrosation of the redox-sensitive conserved

cysteine residues [75]. Interestingly, sensitivity to nitro-

sation could be attenuated by both treatment with dithio-

threitol, as well as the thioredoxin system [75].

Matrix metalloproteases

Matrix metalloproteases (MMPs) have been implicated

in the pathogenesis of stroke, neurodegenerative disease and

cellular metastasis. Numerous articles point to the modu-

lation of MMP expression and activity by nitric oxide

[30,112,113], and it is believed that MMP-9 expression is

suppressed through the NFKB pathway [76]. Alternately,

Gu et al. have shown that MMP-9 is activated by S-

nitrosation in vitro using an MMP-9 construct complete

with the pro-peptide and catalytic domains but lacking the

hemopexin domain to reduce interfering effects of tissue

inhibitors of MMPs [29]. MMP-9 purified from HEK293

cell supernatants and exposed to S-nitrosocysteine showed

significantly increased activity via cleavage of a fluorogenic

substrate I peptide [29]. Mass spectral analysis of the nature

of the interaction between nitric oxide and MMP-9 revealed

that instead of formation of a stable S-nitrosothiol deriva-

tive, a tryptic fragment was obtained that corresponded to

the sulfonic acid derivative of the pro-peptide [29]. This

same fragment was also observed in in vivo immunopreci-

pitated MMP-9 from rat brain subjected to 2 h cerebral

ischaemia with 15 min reperfusion versus control [29]. Of

the 19 available cysteines found in MMP-9, only the

cysteine in the pro-peptide domain was irreversibly modi-

fied [29]. Further, iNOS inhibition in the same experimental

protocol abrogated formation of the modified pro-peptide

cysteine, appearing to confirm the role of nitric oxide in

pathophysiological activation of MMP-9 in ischaemia-

reperfusion injury [29]. Given that the pro-peptide domain

cysteine is strictly conserved in all MMPs and responsible

for maintaining them in an inactive state [71], it is possible

that this mode of activation may be effective in other MMPs

as well.

Viral proteins

SNO and nitrosothiols are reported to modulate the life

cycle of a variety of viruses, including human immunode-

ficiency [61,110], herpes simplex type 1 [12] and Epstein–

Barr viruses [11]. A role forSNO in the pathophysiology of

viral infections is further suggested by reports indicating

over-production of the free radical in HIV-1-infected

patients [98]. Investigators have inquired as to whether the

modulative functions of nitric oxide and nitrosothiols may

be elicited in part through protein S-nitrosation of thiols

critical to virion maturation [5,55,79,87].

Evidence demonstrating an interaction betweenSNO and

cysteine residues of HIV-1 protease (HIV-PR) has been

provided [5,79,87]. HIV-PR possesses proteolytic activity

critical to viral replication [57], cleaving precursor proteins

to generating virion structural proteins and enzymes. HIV-

PR bears two ‘‘relatively conserved’’ cysteine residues

(Cys67 and Cys95) exposed to the viral surface that may

regulate its catalytic activity [87]. It has been shown that

treatment of purified HIV-1 protease with the NO donor

NOR-3 causes a dose-dependent decrease in protease

activity and that inhibition could be overcome by treatment

with thiol reductant dithiothreitol [79]. Sehajpal et al.

elaborated on these results using purified recombinant

HIV-PR exposed to nitric oxide generated from sodium

nitroprusside, S-nitrosoacetylpenicillamine and iNOS [87].

A concentration-dependent decrease in proteolytic activity

was observed upon exposure toSNO that could also be

restored by treatment with reducing agent [87]. Treatment of

HIV-PR withSNO resulted in the observation of both 30 and

60 Da shifted species in the monomer and 60 and 120 Da

shifted species in the dimer by electrospray ionization mass

spectrometry [87]. To confirm that addition of theSNO

moiety was occurring at the available cysteine residues,

thiols were blocked with N-ethylmaleimide (NEM). As

expected, two moles and four moles of the thiol-blocking

agent added to the HIV-PR monomer and dimer, respec-

tively, and effectively blocked formation of the 30-, 60- and

120 Da-shifted species [87]. Interestingly, the authors noted

the absence of a 45-Da species generally indicative tyrosine

of nitration [87].

The ability ofSNO and nitrosothiols to inhibit HIV-1

protease activity has been extended to show inhibition of viral

replication in human cells. Indeed, reports indicate that

nitrosothiols inhibit replication of the HIV-1 virus in acutely

infected peripheral blood mononuclear cells [61] in a dose-

dependent manner. It has also been suggested that nitric oxide

delivered by a variety of NO donors may inhibit HIV-1

reverse transcriptase activity through oxidation of catalytic

cysteine residues [78]. This, however, was not observed

during measurement of reverse transcriptase activity in cell-

free supernatants of HIV-1-infected peripheral blood mono-

nuclear cells in the presence or absence of S-nitrosoacetylpe-

nicillamine [61]. Although, nitric oxide may initially appear

to have anti-viral effects, as testament to the complexity of

cellular signals transmitted by nitric oxide through S-nitro-

sation, it has been shown that inhibition of endogenousSNO

production may serve to assist recovery of the proliferative

response in T cells in AIDS patients [72]. This observation

serves to underscore the importance of reconciling the

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 787

numerous functional apoptotic, proliferative and anti-viral

interactions of nitric oxide in an in vivo setting.

Denitrosation of low molecular weight thiols

Various mechanisms for the decomposition of low

molecular weight thiols have been outlined in the

literature including metal-, chemical- and enzyme-induced

decomposition.

Metal ion-mediated decomposition of nitrosothiols

Cu+ is known to rapidly degrade S-nitrosothiols, such as

S-nitrosocysteine and S-nitrosoglutathione, via reductive

homolysis of the S–NO bond, and this reductive process

can be mimicked by cupric ion in the presence of

glutathione, cysteine or ascorbate [25].

RSNO þ Cuþ Y RS� þ SNOþ Cu2þ ð18Þ

Decomposition can be blocked with the copper(I)-

chelator neocuproine, but not the copper(II)-chelator,

cuprizone, thus emphasizing the major role of Cu+ [25].

Although intracellular and circulating levels of unbound

copper are reported to be undetectable [83], it has been

noted that protein-bound sources of cupric ion are capable

of generatingSNO from nitrosothiols [15].

Superoxide-mediated decomposition of nitrosothiols

Trujillo et al. reported xanthine oxidase-mediated decom-

position of S-nitrosoglutathione and suggested a mechanism

by which enzymatically generated superoxide reduces the

SNO bond yielding oxygen, reduced thiol and nitric oxide

[99]. This reaction, which occurs only in the presence of

oxygen, was partially inhibitable by superoxide dismutase

and was also found to yield peroxynitrite by the fast

secondary reaction of liberatedSNO with available O2

S�

[99]. Estimates of the rate constant for this reaction were

reported as 1.0 � 104 M�1s�1, approximately 105 times

slower than the diffusion-controlled reaction of superoxide

with nitric oxide [99].

Enzyme-mediated decomposition of nitrosothiols

Xanthine oxidase. Xanthine oxidase is a homodimeric,

redox-active enzyme that is capable of utilizing purine or

pteridine substrates to catalyze the reduction of molecular

oxygen forming superoxide [99]. In addition to superoxide-

dependent decomposition S-nitrosothiols, Trujillo et al. also

noted significant denitrosation of cysteine–NO in the

absence of oxygen, indicating that S-nitrosocysteine serves

as an electron acceptor for xanthine oxidase [99]. Interest-

ingly, this observation could not be extrapolated to S-

nitrosoglutathione, in that denitrosation was completely

abolished in the presence of superoxide dismutase [99]. This

disparity is likely due to differences in the reduction

potential between S-nitrosospecies.

Protein disulfide isomerase. The enzyme protein disulfide

isomerase (PDI) is a multifunctional enzyme found primar-

ily in the endoplasmic reticulum, but also localized to the

surface of a variety of cells, including platelets, endothelial

cells and fibroblasts [18,85]. In addition, it appears that this

enzyme may also localize to regions outside additional non-

ER regions of the cell [100]. PDI has been shown to

catalyze the denitrosation of low molecular weight nitro-

sothiols and is itself nitrosatable [111] at its active site thiols.

Recent studies by Sliskovic et al. indicate that PDI catalyzes

the denitrosation of low-molecular weight thiols via a

mechanism that involves a nitroxyl disulfide intermediate

and yields authenticSNO and an oxidized disulfide active

site [91]. In the absence of suitable reducing agents to cycle

it back to the reduced form, oxidized PDI is then rendered

incapable of further denitrosation. Alternately, the oxidized

form is then active in disulfide exchange reactions, thus

providing the basis for a nitric oxide-induced molecular

switch between the various activities of PDI. Most interest-

ingly, this study also showed that native, reduced PDI is

capable of denitrosating nitrosated analogs of itself, thus

liberating nitric oxide. Investigation of other potential

protein targets of PDI denitrosative activity would appear

appropriate.

Cu,Zn superoxide dismutase. Cu,Zn superoxide dismutase

is a copper-bearing enzyme found both intracellularly and

on the periphery of some cells, catalyzing the conversion of

superoxide to peroxide, thus providing a first line of defense

against superoxide-mediated oxidative damage. The ability

of copper ions to efficiently decompose the SNO bond

provided the impetus to investigate the role of this enzyme

in enzymatic decomposition of S-nitrosothiols. It has been

demonstrated that Cu,Zn SOD is capable of denitrosating S-

nitrosoglutathione and that this ability is severely compro-

mised as the concentration of glutathione is increased to

levels found intracellularly [42]. Physiologically, this would

suggest that Cu,Zn SOD-mediated denitrosation is of

limited importance in the cellular interior, however, and

would increase in significance for enzyme localized to the

ectoplasmic surface of the cell where glutathione availability

is limited.

Glutathione-dependent formaldehyde dehydrogenase. Glu-

tathione-dependent formaldehyde dehydrogenase (GS-FDH)

was found to be an efficient GSNO reductase [57].

Homozygous knockout of the GSNO reductase in mice

(GS-FDH�/�) resulted in abrogation of GSNO reductase

activity with a variety of reducing agents compared to wild

type [57]. Levels of protein-bound SNO content were

reportedly increased by 50% in hepatocytes of GS-FDH�/�as compared to littermates [57].

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791788

c-Glutamyl transferase. Studies on the effects of the

ventilatory response to hypoxia in a mouse model revealed

that nitric oxide plays a role in eliciting an increase in

minute ventilation following hypoxia [56]. This response

was found to be stereoselective using the d and l isomers of

S-nitrosocysteine, indicating a role for enzyme-mediated

decomposition of the NO-donating agent [56]. Observed

increases in post-hypoxic ventilation could be inhibited by

pharmacological treatment with acivicin, an inhibitor of g-

glutamyl transferase (g-GT) [56]. A role for this enzyme in

the bioactivation of GSNO was further corroborated using

homozygous g-GT knockout mice in which investigators

found that these mice display an attenuated ventilatory

response to hypoxia [56], likely through an inability to

bioactivate the NO donor. To our knowledge, the deni-

trosation activity of this enzyme has not been characterized

in vitro.

Thioredoxin/thioredoxin reductase. GSNO has been deter-

mined to be a substrate for calf thymus thioredoxin

reductase (TR) in the presence of NADPH with an apparent

KM of 60 AM [74]. Rates of GSNO denitrosation were

further found to increase at lower [GSNO] upon addition of

human thioredoxin to TR but were inhibitory at higher

[GSNO] [74]. Oxidation of Trx active site vicinal thiols was

observed with GSNO, liberating authenticSNO and GSH

[74]. In light of the observed ability of the mammalian

thioredoxin system to liberate nitric oxide from both GSNO

[74], as well as S-nitrosated AP-1 subunits [75], the

denitrosative ability of thioredoxin may extend to SNO

proteins in a broader sense.

Denitrosation of S-nitrosated protein thiol residues

There have recently been reported several accounts of

signals initiated at the plasma membrane that result in

denitrosation of S-nitrosated proteins [35,60]. These

accounts represent some of the first indications of the

dynamic nature of protein S-nitrosylation and suggest that

signals initiated at the plasma membrane can transduce

intracellular signals that modulate protein SNO levels in

similar fashion to phosphorylation/dephosphorylation

events. Studies on the nitrosation status of the pro-

apoptotic protein caspase-3 reveal that Fas activation

appears to induce denitrosation of caspase-3 in human B

and T cells [60]. S-nitrosation was found to occur at the

active-site cysteine by transfecting MCF-7 cells (that do

not express caspase-3) with either wild-type or C Y A

mutants [60]. Measurement ofSNO released by photolysis-

chemiluminescence showed consistently higher levels ofSNO in the wild-type versus mutant caspase-3 [60].

Decreased levels of S-nitrosated caspase-3 were observed

after 1.5–2 h following stimulation of MCF-7 cells using

Fas agonist antibody versus unstimulated cells [60].

Further, inhibition of nitric oxide production with N-g-

monomethyl arginine (l-NMMA) failed to show signifi-

cant change in levels of cysteine nitrosation within 2 h,

thus confirming that the decline in S-nitrosated caspase-3 is

due to denitrosation and not reduced nitrosation [60]. The

influence of the pro-inflammatory and atherogenic sub-

stances, tumor necrosis factor (TNFa) and mildly oxidized

low density lipoprotein (oxLDL), on protein S-nitrosation

status has been investigated in endothelial cells [35]. Both

substances appear to reduce the levels of S-nitrosated

protein in endothelial cell lysates by approximately 50% as

measured by the Griess–Saville assay [35]. The authors

noted SNO content was largely confined to the high

molecular weight lysate fraction, demonstrating minimal

SNO contribution from low molecular weight thiols [35].

Further, it was observation that inhibition of endothelial

nitric oxide synthase with l-NMMA did not reduce protein

SNO levels at 18 h [35]. In contrast, both TNF-a and

oxLDL reduced protein SNO levels to approximately 50%

at 18 h, suggesting again that protein denitrosation is an

active process [35]. Correspondingly, it has been reported

that death signals transmitted by application of TNF-a to

human colon adenocarcinoma cells appear to result in

denitrosation of endogenous procaspase-9, thus contribu-

ting to the zymogen cleavage and execution of the

apoptotic program [46]. These examples appear to repre-

sent the first observation of signals initiated at the plasma

membrane that culminate in changes in the status of

protein S-nitrosation intracellularly; however, molecular

characterization of the species active in denitrosation

remains.

Conclusions

Clearly the paradigm of S-nitrosation as a mode of

transducing transient biological signals is posed to grow in

scope and importance. The ability of S-nitrosation to

modulate protein activity and thus cellular physiology has

been recognized, but how nitrosation alters function is not

always clear [29,104] and may reveal additional interesting

examples. Undoubtedly, elucidation of the interactions

between reactive nitrogen species and protein thiols in the

context of transducing cellular signals will establish the

foundations on which investigators may gain insight into

how pathological changes inSNO metabolism and protein

expression contribute to perturbed communications.

References

[1] Abate C, Patel L, Rauscher III FJ, Curran T. Redox regulation of fos

and jun DNA-binding activity in vitro. Science 1990;249(4973):

1157–61.

[2] Arnelle DR, Stamler JS. NO+, NO, and NO- donation by S-

nitrosothiols: implications for regulation of physiological functions

by S-nitrosylation and acceleration of disulfide formation. Arch

Biochem Biophys 1995;318(2):279–85.

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 789

[3] Ascenzi P, Colasanti M, Persichini T, Muolo M, Polticelli F,

Venturini G, Bordo D, Bolognesi M. Re-evaluation of amino acid

sequence and structural consensus rules for cysteine-nitric oxide

reactivity. Biol Chem 2000;381(7):623–7.

[4] Bartberger MD, Liu W, Ford E, Miranda KM, Switzer C, Fukuto JM,

Farmer PJ, Wink DA, Houk KN. The reduction potential of nitric

oxide (NO) and its importance to NO biochemistry. Proc Natl Acad

Sci U S A 2002;99(17):10958–63.

[5] Basu A, Sehajpal PK, Ogiste JS, Lander HM. Targeting cysteine

residues of human immunodeficiency virus type 1 protease by

reactive free radical species. Antioxid Redox Signal 1999;1(1):

105–12.

[6] Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA.

Apparent hydroxyl radical production by peroxynitrite: implications

for endothelial injury from nitric oxide and superoxide. Proc Natl

Acad Sci U S A 1990;87(4):1620–4.

[7] Boullerne AI, Rodriguez JJ, Touil T, et al. Anti-S-nitrosocysteine

antibodies are a predictive marker for demyelination in experimental

autoimmune encephalomyelitis: implications for multiple sclerosis.

J Neurosci 2002;22(1):123–32.

[8] Broillet MC. S-nitrosylation of proteins. Cell Mol Life Sci 1999;

55(8–9):1036–42.

[9] Bryan NS, Rassaf T, Maloney RE, et al. Cellular targets and

mechanisms of nitros(yl)ation: an insight into their nature and

kinetics in vivo. Proc Natl Acad Sci U S A 2004;101(12):

4308–13.

[10] Ckless K, Reynaert NL, Taatjes DJ, Lounsbury KM, van der Vliet A,

Janssen-Heininger Y. In situ detection and visualization of S-

nitrosylated proteins following chemical derivatization: identification

of Ran GTPase as a target for S-nitrosylation. Nitric Oxide 2004;

11(3):216–27.

[11] Colasanti M, Persichini T, Venturini G, Ascenzi P. S-nitrosylation of

viral proteins: molecular bases for antiviral effect of nitric oxide.

IUBMB Life 1999;48(1):25–31.

[12] Croen KD. Evidence for antiviral effect of nitric oxide. Inhibition of

herpes simplex virus type 1 replication. J Clin Invest 1993;91(6):

2446–52.

[13] delaTorre A, Schroeder RA, Punzalan C, Kuo PC. Endotoxin-

mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene

transcription in ANA-1 murine macrophages. J Immunol 1999;

162(7):4101–8.

[14] delaTorre A, Schroeder RA, Kuo PC. Alteration of NF-nB p50 DNA

binnding kinetics by S-nitrosylation. Biochem Biophys Res Commun

1997;238(3):703–6.

[15] Dicks AP, Williams DL. Generation of nitric oxide from S-

nitrosothiols using protein-bound Cu2+ sources. Chem Biol 1996;

3(8):655–9.

[16] Dimmeler S, Haendeler J, Nehls M, Zeiher AM. Suppression of

apoptosis by nitric oxide via inhibition of interleukin-1beta-convert-

ing enzyme (ICE)-like and cysteine protease protein (CPP)-32-like

proteases. J Exp Med 1997;185(4):601–7.

[17] Espey MG, Thomas DD, Miranda KM, Wink DA. Focusing of nitric

oxide-mediated nitrosation and oxidative nitrosylation as a conse-

quence of reaction with superoxide. Proc Natl Acad Sci 2002;99(17):

11127–32.

[18] Essex DW, Chen K, Swiatkowska M. Localization of protein

disulfide isomerase to the external surface of the platelet plasma

membrane. Blood 1995;86(6):2168–73.

[19] Ferrari DM, Soling HD. The protein disulphide-isomerase

family: unravelling a string of folds. Biochem J 1999;339(Pt. 1):

1–10.

[20] Foster MW, McMahon TJ, Stamler JS. S-nitrosylation in health and

disease. Trends Mol Med 2003;9(4):160–8.

[21] Foster MW, Stamler JS. New insights into protein S-nitrosylation.

Mitochondria as a model system. J Biol Chem 2004;279(24):

25891–7.

[22] Fukuto JM, Hobbs AJ, Ignarro LJ. Conversion of nitroxyl (HNO) to

nitric oxide (NO) in biological systems: the role of physiological

oxidants and relevance to the biological activity of HNO. Biochem

Biophys Res Commun 1993;196(2):707–13.

[23] Fukuto JM, Cho JY, Switzer C. Nitric oxide: biology and

pathobiology. 1st ed. San Diego’ Elsevier Science and Technology

Books; 2000.

[24] Gaston BM, Carver J, Doctor A, Palmer LA. S-nitrosylation

signaling in cell biology. Mol Interv 2003 (Aug);3(5):253–63.

[25] Gorren AC, Schrammel A, Schmidt K, Mayer B. Decomposition of

S-nitrosoglutathione in the presence of copper ions and glutathione.

Arch Biochem Biophys 1996;330(2):219–28.

[26] Gow AJ, Buerk DG, Ischiropoulos H. A novel reaction mechanism

for the formation of S-nitrosothiol in vivo. J Biol Chem 1997;

272(5):2841–5.

[27] Gow AJ, Davis CW, Munson D, Ischiropoulos H. Immunohisto-

chemical detection of S-nitrosylated proteins. Methods Mol Biol

2004;279:167–72.

[28] Greenberg CS, Birckbichler PJ, Rice RH. Transglutaminases: multi-

functional cross-linking enzymes that stabilize tissues. FASEB J

1991;5(15):3071–7.

[29] Gu Z, Kaul M, Yan B, et al. S-nitrosylation of matrix metal-

loproteinases: signaling pathway to neuronal cell death. Science

2002;297(5584):1186–90.

[30] Gurjar MV, DeLeon J, Sharma RV, Bhalla RC. Mechanism of

inhibition of matrix metalloproteinase-9 induction by NO in vascular

smooth muscle cells. J Appl Physiol 2001;91(3):1380–6.

[31] Haendeler J, Hoffmann J, Tischler V, Berk BC, Zeiher AM,

Dimmeler S. Redox regulatory and anti-apoptotic functions of

thioredoxin depend on S-nitrosylation at cysteine 69. Nat Cell Biol

2002;4(10):743–9.

[32] Haynes V, Elfering S, Traaseth N, Giulivi C. Mitochondrial nitric-

oxide synthase: enzyme expression, characterization, and regulation.

J Bioenerg Biomembr 2004;36(4):341–6.

[33] Herold S, Rock G. Reactions of deoxy-, oxy-, and methemoglobin

with nitrogen monoxide. Mechanistic studies of the S-nitrosothiol

formation under different mixing conditions. J Biol Chem 2003;

278(9):6623–34.

[34] Hobbs AJ, Fukuto JM, Ignarro LJ. Formation of free nitric oxide

from l-arginine by nitric oxide synthase: direct enhancement of

generation by superoxide dismutase. Proc Natl Acad Sci 2004;

91(23):10992–6.

[35] Hoffmann J, Haendeler J, Zeiher AM, Dimmeler S. TNFalpha and

oxLDL reduce protein S-nitrosylation in endothelial cells. J Biol

Chem 2001;276(44):41383–7.

[36] Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem

1989;264(24):13963–6.

[37] Inoue K, Akaike T, Miyamoto Y, et al. Nitrosothiol formation

catalyzed by ceruloplasmin. Implication for cytoprotective mecha-

nism in vivo. J Biol Chem 1999;274(38):27069–75.

[38] Jaffrey SR, Snyder SH. The biotin switch method for the detection of

S-nitrosylated proteins. Sci STKE 2001;2001(86):PL1.

[39] Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH.

Protein S-nitrosylation: a physiological signal for neuronal nitric

oxide. Nat Cell Biol 2001;3(2):193–7.

[40] Jaffrey SR, Fang M, Snyder SH. Nitrosopeptide mapping: a novel

methodology reveals S-nitrosylation of dexras1 on a single cysteine

residue. Chem Biol 2002;9(12):1329–35.

[41] Jia L, Bonaventura C, Bonaventura J, Stamler JS. S-nitrosohaemo-

globin: a dynamic activity of blood involved in vascular control.

Nature 1996;380(6571):221–6.

[42] Jourd’heuil D, Laroux FS, Miles AM, Wink DA, Grisham MB.

Effect of superoxide dismutase on the stability of S-nitrosothiols.

Arch Biochem Biophys 1999 (Jan 15);361(2):323–30.

[43] Kawashima S. The two faces of endothelial nitric oxide synthase

in the pathophysiology of atherosclerosis. Endothelium 2004;

11(2):99–107.

[44] Keshive M, Singh S, Wishnok JS, Tannenbaum SR, Deen WM.

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791790

Kinetics of S-nitrosation of thiols in nitric oxide solutions. Chem Res

Toxicol 1996;9(6):988–93.

[45] Kim WK, Choi YB, Rayudu PV, et al. Attenuation of NMDA

receptor activity and neurotoxicity by nitroxyl anion, NO. Neuron

1999;24(2):461–9.

[46] Kim JE, Tannenbaum SR. S-nitrosation regulates the activation of

endogenous procaspase-9 in HT-29 human colon carcinoma cells.

J Biol Chem 2004;279(11):9758–64.

[47] Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, Nagano T.

Fluorescent indicators for imaging nitric oxide production. Angew

Chem Int Ed Engl 1999;38(21):3209–12.

[48] Kostka P. Free radicals (nitric oxide). Anal Chem 1995;67(12):

411R–6R.

[49] Kuncewicz T, Sheta EA, Goldknopf IL, Kone BC. Proteomic

analysis reveals novel protein targets of S-nitrosylation in mesangial

cells. Contrib Nephrol 2004;141:221–30.

[50] Lai TS, Hausladen A, Slaughter TF, Eu JP, Stamler JS, Greenberg

CS. Calcium regulates S-nitrosylation, denitrosylation, and activity

of tissue transglutaminase. Biochemistry 2001;40(16):4904–10.

[51] Lander HM, Ogiste JS, Teng KK, Novogrodsky A. p21ras as a

common signaling target of reactive free radicals and cellular redox

stress. J Biol Chem 1995;270(36):21195–8.

[52] Lander HM, Ogiste JS, Pearce SF, Levi R, Novogrodsky A. Nitric

oxide-stimulated guanine nucleotide exchange on p21ras. J Biol

Chem 1995;270(13):7017–20.

[53] Lander HM, Hajjar DP, Hempstead BL, et al. A molecular redox

switch on p21(ras). Structural basis for the nitric oxide-p21(ras)

interaction. J Biol Chem 1997;272(7):4323–6.

[54] Lane P, Hao G, Gross SS. S-nitrosylation is emerging as a specific

and fundamental posttranslational protein modification: head-to-head

comparison with O-phosphorylation. Sci STKE 2000;(86):RE1.

[55] Lebon F, Ledecq M. Approaches to the design of effective HIV-1

protease inhibitors. Curr Med Chem 2000;7(4):455–77.

[56] Lipton AJ, Johnson MA, Macdonald T, Lieberman MW, Gozal D,

Gaston B. S-nitrosothiols signal the ventilatory response to hypoxia.

Nature 2001;413(6852):171–4.

[57] Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS. A

metabolic enzyme for S-nitrosothiol conserved from bacteria to

humans. Nature 2001;410(6827):490–4.

[58] Liu X, Miller MJ, Joshi MS, Thomas DD, Lancaster Jr JR.

Accelerated reaction of nitric oxide with O2 within the hydrophobic

interior of biological membranes. Proc Natl Acad Sci 1998;

95(5):2175–9.

[59] Maejima Y, Adachi S, Morikawa K, Ito H, Isobe M. Nitric oxide

inhibits myocardial apoptosis by preventing caspase-3 activity via S-

nitrosylation. J Mol Cell Cardiol 2005;38(1):163–74.

[60] Mannick JB, Hausladen A, Liu L, et al. Fas-induced caspase

denitrosylation. Science 1999;284(5414):651–4.

[61] Mannick JB, Stamler JS, Teng E, et al. Nitric oxide modulates HIV-1

replication. J Acquir Immune Defic Syndr 1999;22(1):1–9.

[62] Mannick JB, Schonhoff C, Papeta N, et al. S-nitrosylation of

mitochondrial caspases. J Cell Biol 2001;154(6):1111–6.

[63] Marshall HE, Merchant K, Stamler JS. Nitrosation and oxidation in

the regulation of gene expression. FASEB J 2000;14(13):1889–900.

[64] Marshall HE, Stamler JS. Inhibition of NF-kappa B by S-nitro-

sylation. Biochemistry 2001;40(6):1688–93.

[65] Marshall HE, Hess DT, Stamler JS. S-nitrosylation: physiological

regulation of NF-kappaB. Proc Natl Acad Sci U S A 2004;

101(24):8841–2.

[66] Martinez-Ruiz A, Lamas S. S-nitrosylation: a potential new paradigm

in signal transduction. Cardiovasc Res 2004;62(1):43–52.

[67] Matthews JR, Kaszubska W, Turcatti G, Wells TN, Hay RT. Role of

cysteine62 in DNA recognition by the P50 subunit of NF-kappa B.

Nucleic Acids Res 1993;21(8):1727–34.

[68] Matthews JR, Botting CH, Panico M, Morris HR, Hay RT. Inhibition

of NF-kappaB DNA binding by nitric oxide. Nucleic Acids Res

1996;24(12):2236–42.

[69] Melino G, Bernassola F, Knight RA, Corasaniti MT, Nistico G,

Finazzi-Agro A. S-nitrosylation regulates apoptosis. Nature 1997;

388(6641):432–3.

[70] Miranda KM, Paolocci N, Katori T, et al. A biochemical rationale for

the discrete behavior of nitroxyl and nitric oxide in the cardiovascular

system. Proc Natl Acad Sci U S A 2003;100(16):9196–201.

[71] Morgunova E, Tuuttila A, Bergmann U, Tryggvason K. Structural

insight into the complex formation of latent matrix metalloproteinase

2 with tissue inhibitor of metalloproteinase 2. Proc Natl Acad Sci

2002;99(11):7414–9.

[72] Mossalayi MD, Becherel PA, Debre P. Critical role of nitric oxide

during the apoptosis of peripheral blood leukocytes from patients

with AIDS. Mol Med 1999;5(12):812–9.

[73] Murphy ME, Sies H. Reversible conversion of nitroxyl anion to nitric

oxide by superoxide dismutase. Proc Natl Acad Sci 1991;88(23):

10860–4.

[74] Nikitovic D, Holmgren A. S-nitrosoglutathione is cleaved by the

thioredoxin system with liberation of glutathione and redox regulat-

ing nitric oxide. J Biol Chem 1996;271(32):19180–5.

[75] Nikitovic D, Holmgren A, Spyrou G. Inhibition of AP-1 DNA

binding by nitric oxide involving conserved cysteine residues in Jun

and Fos. Biochem Biophys Res Commun 1998;242(1):109–12.

[76] Okamoto T, Akuta T, Tamura F, van Der Vliet A, Akaike T.

Molecular mechanism for activation and regulation of matrix metal-

loproteinases during bacterial infections and respiratory inflamma-

tion. Biol Chem 2004;385(11):997–1006.

[77] Park SK, Lin HL, Murphy S. Nitric oxide regulates nitric oxide

synthase-2 gene expression by inhibiting NF-kappaB binding to

DNA. Biochem J 1997;322(Pt 2):609–13.

[78] Persichini T, Colasanti M, Fraziano M, Colizzi V, Ascenzi P, Lauro

GM. Nitric oxide inhibits HIV-1 replication in human astrocytoma

cells. Biochem Biophys Res Commun 1999;254(1):200–2.

[79] Persichini T, Colasanti M, Lauro GM, Ascenzi P. Cysteine nitro-

sylation inactivates the HIV-1 protease. Biochem Biophys Res

Commun 1998;250(3):575–6.

[80] Powis G, Oblong JE, Gasdaska PY, Berggren M, Hill SR, Kirkpatrick

DL. The thioredoxin/thioredoxin reductase redox system and control

of cell growth. Oncol Res 1994;6(10–11):539–44.

[81] Quijano C, Alvarez B, Gatti RM, Augusto O, Radi R. Pathways of

peroxynitrite oxidation of thiol groups. Biochem J 1997;322(Pt. 1):

167–73.

[82] Radi R, Denicola A, Alvarez B, Ferrer-Sueta G, Rubbo H. The

biological chemistry of peroxynitrite. In: Ignarro JL, editor. Nitric

oxide: biology and pathobiology. New York’ Academic Press; 2000.

p. 57–82.

[83] Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV.

Undetectable intracellular free copper: the requirement of a

copper chaperone for superoxide dismutase. Science 1999;284(5415):

805–8.

[84] Rafikova O, Rafikov R, Nudler E. Catalysis of S-nitrosothiols

formation by serum albumin: the mechanism and implication in

vascular control. Proc Natl Acad Sci U S A 2002;99(9):5913–8.

[85] Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Mechanism

of transfer of NO from extracellular S-nitrosothiols into the cytosol

by cell-surface protein disulfide isomerase. Proc Natl Acad Sci U S A

2001;98(17):9539–44.

[86] Rossig L, Fichtlscherer B, Breitschopf K, et al. Nitric oxide

inhibits caspase-3 by S-nitrosation in vivo. J Biol Chem 1999;274(11):

6823–6.

[87] Sehajpal PK, Basu A, Ogiste JS, Lander HM. Reversible S-

nitrosation and inhibition of HIV-1 protease. Biochemistry 1999;

38(40):13407–13.

[88] Sekkai D, Aillet F, Israel N, Lepoivre M. Inhibition of NF-kappaB

and HIV-1 long terminal repeat transcriptional activation by

inducible nitric oxide synthase 2 activity. J Biol Chem 1998;273(7):

3895–900.

[89] Shin WS, Hong YH, Peng HB, De Caterina R, Libby P, Liao JK.

S. Miersch, B. Mutus / Clinical Biochemistry 38 (2005) 777–791 791

Nitric oxide attenuates vascular smooth muscle cell activation by

interferon-gamma. The role of constitutive NF-kappa B activity.

J Biol Chem 1996;271(19):11317–24.

[90] Shishodia S, Aggarwal BB. Nuclear factor-kappaB: a friend or a foe

in cancer? Biochem Pharmacol 2004;68(6):1071–80.

[91] Sliskovic I, Raturi A, Mutus B. Characterization of the S-denitrosa-

tion activity of protein-disulfide isomerase. J Biol Chem 2005;

280(10):8733–41.

[92] Stamler JS, Toone EJ, Lipton SA, Sucher NJ. (S)NO signals:

translocation, regulation, and a consensus motif. Neuron 1997;

18(5):691–6.

[93] Stamler JS, Jia L, Eu JP, et al. Blood flow regulation by S-

nitrosohemoglobin in the physiological oxygen gradient. Science

1997;276(5321):2034–7.

[94] Stubauer G, Giuffre A, Sarti P. Mechanism of S-nitrosothiol

formation and degradation mediated by copper ions. J Biol Chem

1999;274(40):28128–33.

[95] Sumbayev VV, Budde A, Zhou J, Brune B. HIF-1 alpha protein as a

target for S-nitrosation. FEBS Lett 2003;535(1–3):106–12.

[96] Sumbayev VV. S-nitrosylation of thioredoxin mediates activation of

apoptosis signal-regulating kinase 1. Arch Biochem Biophys 2003;

415(1):133–6.

[97] Tabuchi A, Sano K, Oh E, Tsuchiya T, Tsuda M. Modulation of AP-1

activity by nitric oxide (NO) in vitro: NO-mediated modulation of

AP-1. FEBS Lett 1994;351(1):123–7.

[98] Torre D, Pugliese A, Speranza F. Role of nitric oxide in HIV-1

infection: friend or foe? Lancet Infect Dis 2002;2(5):273–80.

[99] Trujillo M, Alvarez MN, Peluffo G, Freeman BA, Radi R. Xanthine

oxidase-mediated decomposition of S-nitrosothiols. J Biol Chem

1998;273(14):7828–34.

[100] Turano C, Coppari S, Altieri F, Ferraro A. Proteins of the PDI family:

unpredicted non-ER locations and functions. J Cell Physiol 2002;

193(2):154–63.

[101] van der Vliet A, Hoen PA, Wong PS, Bast A, Cross CE. Formation of

S-nitrosothiols via direct nucleophilic nitrosation of thiols by

peroxynitrite with elimination of hydrogen peroxide. J Biol Chem

1998;273(46):30255–62.

[102] Viner RI, Williams TD, Schoneich C. Peroxynitrite modification of

protein thiols: oxidation, nitrosylation, and S-glutathiolation of

functionally important cysteine residue(s) in the sarcoplasmic

reticulum Ca-ATPase. Biochemistry 1999;38(38):12408–15.

[103] Wade RS, Castro CE. Redox reactivity of iron(III) porphyrins and

heme proteins with nitric oxide. Nitrosyl transfer to carbon, oxygen,

nitrogen, and sulfur. Chem Res Toxicol 1990;3(4):289–91.

[104] Williams JG, Pappu K, Campbell SL. Structural and biochemical

studies of p21Ras S-nitrosylation and nitric oxide-mediated guanine

nucleotide exchange. Proc Natl Acad Sci U S A 2003;100(11):

6376–81.

[105] Wink DA, Nims RW, Darbyshire JF, et al. Reaction kinetics for

nitrosation of cysteine and glutathione in aerobic nitric oxide

solutions at neutral pH. Insights into the fate and physiological

effects of intermediates generated in the NO/O2 reaction. Chem Res

Toxicol 1994;7(4):519–25.

[106] Wong PS, Hyun J, Fukuto JM, et al. Reaction between S-nitro-

sothiols and thiols: generation of nitroxyl (HNO) and subsequent

chemistry. Biochemistry 1998;37(16):5362–71.

[107] Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-

kappa B/Rel in induction of nitric oxide synthase. J Biol Chem 1994;

269(7):4705–8.

[108] Yang Y, Loscalzo J. S-nitrosoprotein formation and localization in

endothelial cells. Proc Natl Acad Sci 2005;102(1):117–22.

[109] Yasinska IM, Kozhukhar AV, Sumbayev VV. S-nitrosation of

thioredoxin in the nitrogen monoxide/superoxide system activates

apoptosis signal-regulating kinase 1. Arch Biochem Biophys 2004;

428(2):198–203.

[110] Yasinska IM, Sumbayev VV. S-nitrosation of Cys-800 of HIF-1alpha

protein activates its interaction with p300 and stimulates its tran-

scriptional activity. FEBS Lett 2003;549(1–3):105–9.

[111] Zai A, Rudd MA, Scribner AW, Loscalzo J. Cell-surface protein

disulfide isomerase catalyzes transnitrosation and regulates intra-

cellular transfer of nitric oxide. J Clin Invest 1999;103(3):393–9.

[112] Zaragoza C, Balbin M, Lopez-Otin C, Lamas S. Nitric oxide

regulates matrix metalloprotease-13 expression and activity in

endothelium. Kidney Int 2002;61(3):804–8.

[113] Zhang X, Wang HM, Lin HY, Liu GY, Li QL, Zhu C. Regulation of

matrix metalloproteinases (MMPS) and their inhibitors (TIMPS)

during mouse peri-implantation: role of nitric oxide. Placenta 2004;

25(4):243–52.