Biochimica et Biophysica Acta 1411 (1999) 385^400

Review

Biological aspects of reactive nitrogen species

Rakesh P. Patel b , Joanne McAndrew b , Hassan Sellak b , C. Roger White
Hanjoong Jo b , Bruce A. Freeman a;d , Victor M. Darley-Usmar a;b; *

a;c

Center for Free Radical Biology, University of Alabama at Birmingham, Volker Hall Room GO38, 1670 University Boulevard,
Birmingham, AL 35294-0019, USA
b
Department of Pathology, University of Alabama at Birmingham, Volker Hall Room GO38, 1670 University Boulevard, Birmingham,
AL 35294-0019, USA
c
Departments of Medicine, Vascular Biology and Hypertension, University of Alabama at Birmingham, 619 19th Street South, THT 940,
Birmingham, AL 35233, USA
d
Department of Anesthesiology, University of Birmingham at Alabama, 619 19th Street South, THT 940, Birmingham, AL 35233, USA
Received 6 July 1998; received in revised form 13 October 1998; accepted 9 November 1998

Abstract
Nitric oxide (NO) plays an important role as a cell-signalling molecule, anti-infective agent and, as most recently
recognised, an antioxidant. The metabolic fate of NO gives rise to a further series of compounds, collectively known as the
reactive nitrogen species (RNS), which possess their own unique characteristics. In this review we discuss this emerging aspect
of the NO field in the context of the formation of the RNS and what is known about their effects on biological systems. While
much of the insight into the RNS has been gained from the extensive chemical characterisation of these species, to reveal
biological consequences this approach must be complemented by direct measures of physiological function. Although we do
not know the consequences of many of the dominant chemical reactions of RNS an intriguing aspect is now emerging. This
review will illustrate how, when specificity and amplification through cell signalling mechanisms are taken into account, the
less significant reactions, in terms of yield or rates, can explain many of the biological responses of exposure of cells or
physiological systems to RNS. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Nitric oxide; Superoxide; Peroxynitrite; Nitrotyrosine ; Apoptosis; Cell signaling; Heme protein; Inammation

Contents
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

386

2.

Formation of reactive nitrogen species in biological systems . . . . . . . . . . . . . . . . . . . . . . .

386

3.

Reactive nitrogen species in membranes and lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . .

388

Abbreviations: GTP, guanosine triphosphate; HOCl, hypochlorous acid; LDL, low density lipoprotein; MAP kinase, mitogen acti
vated protein kinase; NO, nitric oxide; NO2 , nitrogen dioxide radical; N2 O4 , dinitrogen tetroxide; NO3
2 , nitrite; NO2 , nitronium cation;
3
3
NOS, nitric oxide synthase; O2 , superoxide anion radical; ONOO , peroxynitrite; PARP, poly(ADP) ribose polymerase; RNS, reactive
nitrogen species
* Corresponding author, at address b. Fax: (205) 9341775; E-mail : darley@path.uab.edu
0005-2728 / 99 / $ ^ see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 5 - 2 7 2 8 ( 9 9 ) 0 0 0 2 8 - 6

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4.

Scavenging peroxynitrite reaction with metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

389

5.

Nitration at sites of inammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

390

6.

RNS and cell signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

392

7.

Peroxynitrite and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

392

8.

Reactions of RNS to yield NO donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

394

9.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

395

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

395

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

395

1. Introduction
The intensive research surrounding nitric oxide
(NO) and its metabolites has given rise to a new
term, the `reactive nitrogen species' (RNS), in the
biological literature [1^3]. By strict chemical criteria
`RNS' encompasses such a diverse range of compounds, with contrasting and distinct properties,
that their only unifying characteristic can be derivation from NO. However, recent advances are giving
important insights into the biology and biochemistry
of specic RNS, their eects on normal physiology
and potential contribution to disease. An aspect we
will dissect here are the reactions with other free
radicals or oxygen to generate the RNS and their
biological eects (Fig. 1).
Intense investigation over the last 10 years has led
to a rapid change in the general concepts relating to
free radical biochemistry. It is now clear that free
radicals are not necessarily short lived and reactive,
but like any other class of compounds exhibit a
broad range of chemical and physical properties.
Likewise their biological properties are not always
deleterious. An interesting and topical example is
nitric oxide, which is relatively specic in its eects,
not very reactive and serves important physiological
functions. Like reactive oxygen species (ROS), RNS
are derived from the interactions of biologically generated free radicals to form more persistent species
resulting in multiple biological eects.
Nitric oxide is formed by a family of enzymes, and
directly serves signalling functions in the cell. Nitric
oxide plays an essential part of normal physiology,

discussed in detail in the other articles in this issue,

and is a potential route to the pathophysiology associated with a number of diseases. From one perspective the most signicant reactions of RNS are those
which occur with the highest yield. However, since
cells have the capacity to amplify signals and select
specic chemical species, then the reactions of RNS
of most physiological or pathological signicance can
only be determined by examining their eects in a
biological system. Reactions of NO ultimately lead
to the oxidation, nitration (addition of NO2 ), nitrosation (addition of NO ), nitrosylation (NO) of most
classes of biomolecules. One of the best known interactions of NO leading to cell signalling is the reversible covalent binding, nitrosylation, with the ferrous
haem in soluble guanylate cyclase. This aspect of NO
signalling will be discussed in detail in other articles
in this volume. Here we will discuss the reactions of
RNS derived from NO with proteins, lipids and carbohydrates.
2. Formation of reactive nitrogen species in biological
systems
In biological systems the primary source of all
RNS is NO (Fig. 1). The biochemistry of NO formation has been discussed in depth in this issue but a
key factor relevant to this discussion is the range of
concentrations of NO (20 nM^2 WM) generated by
the nitric oxide synthases (NOS) [1,2]. However, this
factor places some constraints on the mechanisms of
RNS formation since the maximal activation of these

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Fig. 1. Nitric oxide-dependent reactions that lead to the formation of RNS. Through the rapid reaction of NO and O3
2 , peroxynitrite (ONOOH) is formed and leads to oxidation, nitrosation (addition of NO) or nitration (addition of NO2 ). Nitric
oxide is formed from the enzymatic action of the nitric oxide
synthases (NOS) which under some circumstances can also pro3
duce O3
2 . There are further potential sources of O2 in the cell
including xanthine oxidase (XOD), NAD(P)H oxidases
(NAD(P)H ox and respiratory complexes within the mitochondria (Mito). Recently it has also been shown that the enzyme
myeloperoxidase (MPO) can cause nitration reactions using nitrite (NO3
2 ) and its product hypochlorite (HOCl) as a substrate.
The oxidation reactions (e.g. oxidation of glutathione) occur at
the highest chemical yield but the nitration (e.g. formation of
nitrotyrosine) or nitrosation (e.g. formation of S-nitrosoglutathione) reactions are also biologically signicant since they can
alter cell signalling and generate NO donors.

enzymes is unlikely to yield steady state concentrations of NO higher than 1^2 WM in the cytosolic
compartment. The most prominent NO reactions
which occur at these relatively low concentrations
are with the transition metal centres, iron and copper, in proteins or reactions with other free radicals
and oxygen. The reactions of NO with iron centres
are well established but that with copper containing
proteins is still emerging. One of the most signicant
in biological terms is likely to be the reaction of NO
with the copper in the oxygen binding site of cytochrome c oxidase (fully discussed in an accompanying article).
The rapid reactions of NO with free radicals has
emerged as one of the major routes to the formation
of RNS and, at present, the best understood of these
is the reaction with superoxide (O3
2 ) to form peroxynitrite (ONOO3 ) [4,5]. Peroxynitrite is chemically
unstable under physiological conditions resulting in

387

the formation of nitrate through isomerisation. Since

nitrate is essentially biochemically inert in mammalian cells then this reaction was viewed as an excellent method to scavenge and neutralise O3
2 [6]. As
studies on this interaction progressed a new perspective emerged as it was realised that ONOO3 is reactive with all the major classes of biomolecules and,
therefore, has the potential to mediate cytotoxicity
independently of NO or O3
2 [4].
While the sources of NO are mostly restricted to
the NOS isozymes other metabolic pathways, involving nitrite, are now emerging, particularly under
acidic or ischaemic conditions, as discussed in the
accompanying article by Dr. J. Zweier. The production of O3
2 appears to arise from diverse candidates
that give variable and poorly dened rates of O3
2
production [7] (Fig. 1). Under some circumstances,
such as exposure to high concentrations of lipoproteins or in the presence of redox cycling xenobiotics,
the electron transfer between NAD(P)H in NOS and
arginine becomes `uncoupled' resulting in the accelerated formation of O3
2 [8]. This has led to the provocative idea that in these situations NOS can serve
as an ONOO3 synthase [9]. Peroxynitrite can mediate one electron transfer reactions that produce a
free radical species. In a lipid this can result in initiation of a self-sustaining peroxidation reaction
[10,11]. Peroxynitrite-dependent two electron oxidations can modify molecules with aromatic structures
such as the important antioxidant K-tocopherol [11].
In this case a free radical is not formed from either
ONOO3 or the reactant.
It was demonstrated early on that ONOO3 can
react with both low molecular weight and protein
bound thiols [12^15]. This results in formation of
thiyl radicals and leads to thiol oxidation. Based on
the high intracellular concentrations of thiols it has
been argued that the reaction between ONOO3 and
glutathione represents a major protection mechanism
against ONOO3 -dependent oxidative injury [13],
although under some circumstances thiols can exacerbate damage [16]. Furthermore, this reaction also
results in the formation of S-nitrosoglutathione, an
NO donating molecule, and together with modication of the redox status of the cell could have important implications in a variety of cell signalling
pathways [17^19]. While this reaction occurs at a
low yield the intracellular metabolic release of NO

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is likely to be cytoprotective through both its functions as a cell signalling molecule and antioxidant.
Nitrosation and nitration reactions produce chemically stable derivatives and are able to elicit biological eects distinct from their progenitors. Initially, it
was thought that the stable nitration of tyrosine was
unique to ONOO3 and therefore a marker for its
formation [20]. Nitrotyrosine antibodies have been
important investigative tools in determining the role
of nitration reactions in NO-dependent pathophysiology [21^26]. However, more recent studies have
shown that the enzyme myeloperoxidase (MPO)
can, under some circumstances, induce nitration of
tyrosine. Since nitrotyrosine formation is often associated with inammation, this is an important aspect
that will also be discussed.
3. Reactive nitrogen species in membranes and
lipoproteins
The reaction of NO with oxygen is emerging as a
possible event in the lipophilic domain of membranes
and lipoproteins. Furthermore, the reactions of NO
are substantially modied by the presence of the
chain propagating lipid peroxyl radicals during lipid
peroxidation. First we discuss the reactions of NO in
a biological membrane in which peroxidation is absent and the dominant reactions are those with oxygen. We will then consider how NO behaves in a
system populated with propagating lipid peroxyl radicals.
Since the rate of reaction of oxygen and NO is
proportional to both oxygen and the square of the
NO concentration, it has been argued that this reaction is too slow to contribute to NO metabolism in
an aqueous compartment [27]. However, this situation changes in membranes and lipoproteins with an
estimated 10-fold increase in the oxygen-dependent
consumption of NO [28]. This increase in rate is directly attributable to the 8^10-fold higher solubility
of NO in the hydrophobic phase of the lipid membranes. Taking into account the relative volume of
the cytosol and membrane structures in a cell, it was
estimated that the velocity of the reaction of NO
with oxygen is 300 times greater in the hydrophobic
environment [28]. It would follow that this should
result in higher yields of intermediates, including di-

Fig. 2. Reactions of NO within hydrophobic compartments.

Higher concentrations of NO within hydrophobic compartments
may result in its reaction with oxygen forming a variety of intermediates (e.g. NO2 ) capable of initiating lipid peroxidation
reactions which are propagated by lipid peroxyl radicals (LOO).
Oxidative damage to lipids mediated by NO2 or other oxidant
species may be prevented, however, by termination of propagating peroxyl radicals (LOO) by NO.

nitrogen trioxide (N2 O3 ), dinitrogen tetroxide (N2 O4 )

and the free radical nitrogen dioxide (NO2 ) [29].
While NO2 is an initiator of lipid oxidation [30],
exposure of lipids to NO alone does not result in
lipid peroxidation [31]. As discussed elsewhere in
this issue, NO itself inhibits lipid oxidation induced
by a variety of oxidants [31^33]. An explanation for
this phenomenon is the facile scavenging of lipid peroxyl radicals (LOO) by NO [34], thus preventing signicant accumulation of products of chain propagation (Fig. 2).
The reaction of NO with peroxyl radicals is a further route to lipid derived RNS. Nitric oxide inhibits
oxidative modication of low-density lipoprotein
(LDL), model lipid systems and attenuates the consequent formation of secondary products of lipid oxidation [31^33]. In comparison to other antioxidants,
such as K-tocopherol, NO is extremely ecient even
though two NO molecules are consumed for each
peroxyl radical [33]. This can be explained on the
basis of the rapid rate of the initial termination reaction of NO with peroxyl radicals and because NO
concentrates in lipophilic environments. The termination reaction between NO and LOO will also yield
a transient organic ONOO3 intermediate (LOONO).
These species are unstable and will rearrange by cage
recombination of the alkoxyl/NO2 radical pair to
give primarily an organic nitrate (LONO2 ), diuse
apart to yield alkoxyl/epoxyallylic lipid radical species and NO2 , or hydrolyse to give a lipid alkoxide
and nitrite (NO3
2 ) [35]. However, in the presence of
NO, the decomposition products are themselves ter-

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minated, thus resulting in an overall inhibition of

lipid peroxidation [33]. Since in membranes and lipoproteins the main source of lipid oxidation products
come from propagation reactions, then NO is an
antioxidant in this context. The subsequent biological fate of nitrated lipids may impact on this interpretation and is a focus of further study of this class
of RNS.
The nitration of lipids has also been detected, both
as potential by-products of NO-dependent inhibition
of lipid peroxidation and the direct reaction of lipid
hydroperoxides with RNS. The precise mechanisms
involved are still being dened. Exposure of lipid
peroxidation systems to continuous uxes of NO
and O3
2 can result in the formation of these species
and recent data suggests that acidied NO3
2 will also
react with lipid peroxides to form stable nitrated
products (unpublished results).
The oxidising potential of ONOO3 is sucient
that it can directly initiate lipid peroxidation reactions by abstracting a hydrogen atom from a polyunsaturated fatty acid and also results in the formation of nitrated lipids [9,10,31]. This has been
demonstrated in both model lipid systems and LDL
[9,10,36^38]. The lipoproteins are particularly interesting as they contain a mixture of targets for
ONOO3 including amino acids, antioxidants and unsaturated lipids, and, furthermore, oxidative damage
to LDL is an important aspect step during atherogenesis [39].
In summary, the reaction between RNS and fatty
acids can result in the formation of both oxidised
lipids as well as nitrated lipid species. The latter derivatives may possess both unique reactivities or have
similar eects to oxidised lipids. Since biological systems can be exquisitely sensitive to receptor mediated
eects this is likely to be a productive area for elucidating the eects of lipid based RNS. For example,
nitrated lipids could elicit cellular responses by acting
as agonists for eicosanoid-like G protein-coupled receptors, or by acting as mediators of signal transduction [40].
4. Scavenging peroxynitrite reaction with metals
In biological systems peroxides (e.g. hydrogen peroxide and lipid hydroperoxides) are detoxied by

389

metalloproteins. Initial studies on the reaction between ONOO3 and haem proteins showed that
ONOO3 can oxidise myeloperoxidase and lactoperoxidase forming a haem oxidation intermediate
called compound II [41]. In this reaction the ground
state of the enzyme (containing iron in the ferric i.e.
Fe3 form), is oxidised to the ferryl form (the iron
oxidation state being Fe4 ). This study also demonstrated that ONOO3 can also mediate two electron
transfer processes as the reaction with horseradish
peroxidase resulted with the initial formation of compound I (i.e. the ferryl oxidation state with an associated protein based or porphyrin based cation radical). Furthermore, these reactions are all mediated
by peroxynitrous acid, are rapid and similar to
changes seen with the classical substrate for these
enzymes, hydrogen peroxide. However, it is not
known whether reaction with ONOO3 led to enzyme
inactivation. It has been reported that in the presence
of a peroxide substrate, peroxidases through the formation of compounds I and II mediate oxidative
damage to biological molecules [42]. The formation
of these oxidation states in haem proteins by
ONOO3 could therefore provide an alternate, and
potential amplication mechanism of oxidative damage.
Peroxynitrite also reacts with haemoglobin. Similar to hydrogen peroxide, addition of ONOO3 to
oxyhaemoglobin (i.e. where the iron is in the physiological ferrous oxidation state) resulted in oxidation
of the haem and formation of methaemoglobin (i.e.
ferric) [43,44]. More recently, it was shown that exposure of red blood cells to ONOO3 also resulted in
the formation of methaemoglobin indicating that reaction with haem proteins, in this system at least, can
compete with alternative targets for ONOO3 e.g.
glutathione [45].
In vitro studies also showed that ONOO3 can nitrate haemoglobin [44], and when high concentrations were added to red blood cells covalent modication of membrane proteins occurred [46,47]. It was
demonstrated that ONOO3 could diuse through
the anion exchange channel and across the red blood
cell membrane [45]. This latter property was also
shown in liposomal systems investigating the reaction
between model porphyrin compounds, containing
manganese or iron as the redox active metal centre,
with ONOO3 [48]. These data led the authors to

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suggest that liposome encapsulated porphyrinic compounds may be an ecient strategy to protect against
ONOO3 related oxidative damage [49]. Thus,
although ONOO3 has a relatively short half-life
under physiological conditions, its rate of diusion
across biological membranes is suciently rapid such
that reactions at distant sites of its formation can
occur.
The relatively high reactivity of ONOO3 has made
the development of specic scavengers dicult.
Based on kinetic grounds, however, metal containing
compounds are the most promising candidates. Recently, the role of selenium compounds as potential
peroxynitrite scavengers were reported [50]. It was
shown that the enzyme glutathione peroxidase which
contains a selenocysteine moiety in its active site,
rapidly reduces peroxynitrite to nitrite and protects
against formation of 3-nitrotyrosine [51]. The classical view of this peroxidase enzyme is that it catalyses
the glutathione-dependent reduction of hydrogen
peroxide and lipid hydroperoxides and thus represents an important antioxidant defence mechanism.
On the basis of these ndings ONOO3 should also
be added to this list. It should be noted that although
a reaction between the reduced form of glutathione
peroxidase and ONOO3 does not eect enzyme activity [51], reaction of ONOO3 with the oxidised
enzyme leads to inhibition [52]. Synthetic analogs
of glutathione peroxidase (e.g. ebselen) have been
shown to also possess a `peroxynitrite reductase' activity, and protect against ONOO3 mediated oxidation and nitration reactions [53,54]. The relatively
high rates of reaction of ONOO3 with metal containing compounds suggests that they may make useful ONOO3 scavengers, and are indeed being currently evaluated as such. These studies also
demonstrate that ONOO3 can rapidly react with
non-haem metal containing proteins. Other examples
include the reaction between ONOO3 and copper
containing proteins such as superoxide dismutase
and caeruloplasmin. In the former case this results
in the formation of a species capable of nitrating
phenolics, and in the latter copper is released from
the protein [20,55].
Recent studies suggest that reactions between
ONOO3 and haem proteins may have physiological
functions. The enzyme prostaglandin synthase catalyses the production of prostaglandins and thrombox-

anes, both important mediators of inammation and

other processes [56]. Peroxynitrite was found to be
an excellent substrate for the enzyme peroxidase activity, thereby stimulating cyclooxygenase activity
and the subsequent formation of prostaglandins
[57]. Furthermore, prostaglandin biosynthesis in
macrophages was inhibited by superoxide dismutase
mimetics, indicating that this may be a mechanism
by which ONOO3 can be linked to pro-inammatory reactions in vivo. As yet, no other peroxidase
enzymes have been identied in which ONOO3 can
act as a substrate, but this remains an intriguing
possibility. It is worth mentioning that while
ONOO3 can clearly oxidise numerous haem compounds, this is not the case for all metal containing
proteins. For example guanylate cyclase catalyses the
nitric oxide mediated formation of cyclic guanosine
monophosphate [58]. Similar to haemoglobin this
contains an iron containing haem group in its active
site, but is not oxidised by ONOO3 [59].
Interestingly, ONOO3 can also inactivate haem
proteins. For example all the NOS isoforms were
irreversibly inhibited by ONOO3 , with IC50 values
between 10 and 20 WM [60]. The mechanism of inactivation was suggested to be oxidation and subsequent destruction of the haem-thiolate catalytic
centre. It should be noted, however, that this mechanism does not operate for all haem-thiolate enzymes
as thromboxane synthase was unaected by
ONOO3 , whereas the structurally similar prostacyclin synthase was inhibited, although this mode of
inactivation was due to tyrosine nitration [61]. Disruption of iron-sulphur centres by ONOO3 can also
lead to inactivation of enzymes such as aconitase
[62^64], thereby linking RNS to alterations in mitochondrial function.
5. Nitration at sites of inammation
Cells that are recruited to sites of injury and initiate inammatory events such as macrophages and
activated neutrophils, produce relatively high concentrations of NO (via the inducible isoform of
NOS). It has been suggested that this leads to an
increase in the formation of ONOO3 which is then
used as part of the arsenal against invading pathogens [65]. Although this is an important host defence

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system, similar reactions, as evidenced by the detection of nitrotyrosine, appear to occur during the development of various pro-inammatory diseases such
as atherosclerosis and rheumatoid arthritis [24^26],
and this has led to the hypothesis that ONOO3 is
an important mediator of such pathologies. Nitration
reactions mediated by ONOO3 are inecient relative
to oxidation reactions. More recently, however, it
was shown that at physiological concentrations of
carbon dioxide, the reactivity of ONOO3 is shifted
towards nitration [66^69]. This has been hypothesised to occur via the formation of the intermediate
nitrosoperoxycarbonate (ONOOCOO3 ) [70].
A recent study has shown that the in vitro nitration of tyrosine in aqueous solution is approx. 4%,
following bolus addition of ONOO3 , with yields falling to approx. 0.07% from ONOO3 produced by the
simultaneous generation of NO and O3
2 [71]. This
presents a problem for the hypothesis that 3-nitrotyrosine in biological systems is a product of
ONOO3 , since the only currently recognised biologically relevant source of ONOO3 is through the reaction of NO and O3
2 . At rst glance, this observation may underscore the idea that other mechanisms
contribute to tyrosine nitration in vivo. These alternative mechanisms are, no doubt, signicant and will
be discussed in more detail later.
However, there are a number of factors that need
to be taken into account before the role of ONOO3
in tyrosine nitration in a biological or pathological
setting is ruled out. (1) The fact that the yields from
nitration of tyrosine from the co-generation of NO
and O3
2 are low is not, a priori, critical since nitrated
proteins may accumulate over many days, or even
weeks, if the turnover or metabolism of the modied
protein is also low. It thus becomes important to
understand the relative eciency of the dierent
mechanisms leading to tyrosine nitration. (2) Nitration of free tyrosine in an aqueous milieu does not
take into account that the reactions in a biological
setting may be promoted through the adventitious
binding of transition metals close to tyrosine residues. In fact, not all tyrosines in a protein are
equally susceptible to nitration, for example neighbouring glutamate residues appear to direct nitration
to specic tyrosine residues [72]. This emphasises the

391

point that the local protein environment inuences

the eciency of tyrosine modication, like many other biochemical mechanisms. (3) Catalysis by metalloproteins, such as superoxide dismutase, promotes
nitration of tyrosine and has been suggested to play a
key role in the pathogenesis of motor neurone disease [73]. Considering these issues, and acknowledging the fact that additional mechanisms of tyrosine
nitration are viable in vivo, ONOO3 remains one of
the best characterised and plausible of these processes.
Another important advance in the study of nitration reactions has been the recent ndings showing
that at sites of neutrophil inltration, other RNS
may result in tyrosine nitration and chlorination
[74]. From a biochemical perspective this is perhaps
not surprising since all that is required to achieve
nitration is the stabilisation of a nitrosonium cation
(NO
2 ) donor or a radical termination reaction with
NO2 . The fact that this can be achieved so readily in
biological systems took some time to appreciate but
is now widely accepted. In principle, this means that
one of the major metabolites of NO, nitrite (NO3
2 ),
by
any
compound
can be converted to NO2 or NO
2
which is a suitable one or two electron acceptor respectively (Fig. 3). An interesting example of this is
the reaction of NO3
2 with hypochlorous acid (HOCl)
which generates NO
2 [75^77] via formation of nitryl
chloride (Cl-NO2 ). In this case both chlorination and
nitration of biological phenolic compounds can occur [74]. Furthermore, it was demonstrated that
NO3
2 can be converted into a nitrating agent, presumably via its reduction to NO2 and/or NO
2 , by
myeloperoxidase [78]. Myeloperoxidase is a haem
protein secreted from activated neutrophils, that
uses hydrogen peroxide to produce hypochlorous
acid (HOCl). Other peroxidase enzymes can also catalyse such reactions [79], as illustrated in Fig. 3. Subsequent studies have indeed shown that activated
neutrophils can nitrate model phenolic compounds
as well as tyrosine residues in proteins, and that
3
this can occur in a NO3
2 - or ONOO -dependent
process [74,79]. These studies clearly suggest that
more than one mechanism of nitrotyrosine formation
may operate in vivo especially at sites of inammation.

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Fig. 3. Peroxidase dependent mechanisms of nitration. Mechanisms of nitration which do not involve ONOO3 include peroxidase mediated oxidation of nitrite (NO3
2 ) to either nitrogen
dioxide radical (NO2 ) or nitronium cation (NO
2 ). Myeloperoxidase is a mammalian peroxidase, which has been shown
to mediate nitration of phenolics utilising NO3
2 as a reducing
substrate as outlined in the gure. The rst step (step 1) involves a two electron process by which the resting state of the
enzyme (i.e. a ferric haem, FeIII ) is oxidised to compound I (i.e.
the ferryl oxidation state with an associated protein or porphyrin radical, Xc -FeIV = O). Compound I can be reduced to compound II (i.e. ferryl haem, FeIV = O) by NO3
2 forming NO2
(step 2). In turn compound II can be reduced back to the
ground state of the enzyme by NO2 yielding NO
2 (step 3). Alternatively, NO3
2 may be formed by a two electron reduction
of compound I directly to the ferric form (step 4). Both NO2
and NO3
2 can nitrate phenolic compounds through radical termination or electrophilic substitution reactions respectively. It
should be noted that NO3
2 may also react with compound II
and NO2 with compound I.

6. RNS and cell signalling

Reactive oxygen species and RNS play an essential
role as signalling intermediates. For example, growth
factors, environmental stress, and cytokines regulate
specic cellular responses through redox-sensitive
signalling pathways [80].
What precisely is meant by `redox' modulation of
cell signalling at a molecular level has yet to be
clearly dened. Indications of likely directions for
this area of research have come from studies showing
that NO attenuates inammatory responses induced
by cytokines in vascular endothelial and smooth
muscle cells [81,82] and these eects appear to be
regulated through modulation of NFUB activity
[83]. Furthermore, NO was shown to activate the
GTP binding protein p21Ras.

One mechanism by which NO regulates Ras/MAP

kinases and the Ras/NFUB pathway is S-nitrosation
of Cys118 on Ras [84^86]. This was conrmed using
site-directed mutagenesis of Cys118 to Ser resulting
in a dominant negative phenotype of Ras, which
when transfected into Jurkat cells completely inhibited NO-dependent activation of MAP kinases [87].
More recently, it was demonstrated that NO stimulates haem oxygenase induction, through mechanisms involving both ONOO3 formation and reaction with intracellular thiols [88]. These data are
consistent with S-nitrosation of cysteine residues acting as a pivotal switch to directly modulate enzymes
in signalling cascades or ion channels [86,89].
Indirect eects on cellular signalling pathways by
RNS could also be mediated by tyrosine nitration.
Indeed nitration of tyrosine by ONOO3 results in an
inhibition of tyrosine phosphorylation [90,91] and
appears to target the modied proteins for degradation [90]. Since numerous signalling pathways depend
on tyrosine phosphorylation, nitration reactions represent an important interface between formation of
RNS and cell function. Alternatively, nitration of
certain proteins, such as glutamine synthetase, can
alter protein function and thus compromise their biological role [91]. Also other studies have demonstrated that nitration of tyrosines is associated with
irreversible inhibition of enzyme function [61]. Another important example of tyrosine nitration leading
to the inactivation of an enzyme that could be implicated in cells signalling is the nitration of the mitochondrial Mn superoxide dismutase [92]. This was
shown to occur during rejection of kidneys and since
the enzyme is situated in the mitochondrial matrix
is likely to be an ONOO3 -dependent process.
Although it has received less attention, nitration of
tryptophan also occurs on reaction with RNS, and
could similarly eect electron transfer proteins [93].
7. Peroxynitrite and apoptosis
Interactions with signalling molecules may eect
the outcome of important biological processes such
as apoptosis. Apoptosis is an evolutionarily conserved, genetically controlled process and is an important event during development and homeostasis.
Disruption in the regulation of apoptosis is thought

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to play a key role in the development of numerous

diseases [94^96]. Apoptotic cell death is characterised
by the appearance of certain morphological alterations to cells such as membrane blebbing, chromatin
condensation, DNA fragmentation and cell shrinkage [97]. The process leads to an orderly breakup
of the cell contents forming apoptotic bodies, which
are then phagocytosed by other cells [98]. Apoptosis
can be initiated by a variety of stimuli, both physiological and non-physiological. These stimuli activate
signalling pathways, most of which converge at a
series of caspase enzymes [98,99] which catalyse the
cleavage of the nuclear lamina component, lamin A,
or the DNA repair enzyme poly(ADP) ribose polymerase (PARP) [100^102]. The cleavage of PARP by
caspases, particularly caspase 3, leads to failure in
the repair of DNA damage and ultimately to apoptosis.
Peroxynitrite has been reported to induce DNA
damage and apoptosis in a variety of cell types
such as thymocytes, cortical cells and HL-60 leukaemia cells [103^105]. Similar concentrations of
ONOO3 to those added to the HL-60 cells did not
induce apoptosis in either endothelial cells or blood
cells suggesting that these have a more ecient defence system against ONOO3 mediated damage than
leukaemia cells [106]. The mechanism by which
ONOO3 induces apoptosis is unclear although subsequent formation of reactive oxygen species has
been suggested [107].
Other mechanisms of cellular injury include an
ONOO3 -dependent increase in DNA strand breakage, which triggers the activation of PARP. This enzyme ADP-ribosylates proteins and then extends the
ADP ribose group into a nucleic acid like polymers,
in the process depleting the cell of NAD . Eventually the depletion of cellular NAD results in mitochondrial dysfunction and necrotic cell death [108].
Such changes have been observed in activated macrophages, which have been shown to produce
ONOO3 [108]. A decrease in the NAD content of
the macrophage, which could be inhibited with the
PARP inhibitor 3-aminobenzamide, and inhibition
of mitochondrial respiration also occurred after macrophage activation [108]. Peroxynitrite induced
PARP activation was demonstrated in vascular
smooth muscle cells and was associated with a suppression of aortic contractility. This observation sug-

393

gests that ONOO3 induced PARP activation may

contribute to a loss of vascular function in conditions such as septic shock [109].
Peroxynitrite-induced apoptosis can be inuenced
either positively or negatively by the presence of
growth factors. Pretreatment of broblast cells with
broblast growth factor (FGF-1) augmented the
amount of apoptosis induced by ONOO3 , but has
no eect on programmed cell death induced by other
reactive oxygen species [110]. The mechanism of this
enhanced apoptotic eect is unclear but seems to
involve an increase in the amount of tyrosine nitration. In contrast pretreatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) protected these cells against ONOO3 -induced apoptosis via a phosphatidylinositol-3-kinase-dependent
pathway [73,111]. Some of the rst evidence consistent with ONOO3 -induced apoptosis in a cellular
setting has recently been obtained in motor neurones
and is thought to be relevant to amylotrophic lateral
sclerosis [112].
A number of pathological conditions that have an
inammatory component have been suggested to
progress through ONOO3 -induced apoptosis. These
conditions include bacterial infections, the onset of
insulin-dependent diabetes mellitus (IDDM) and a
number of neurodegenerative diseases.
Infection of the digestive tract by Helicobacter pylori leads to inammatory bowel disease, a risk factor
for gastric cancer [113]. Infection of gastric epithelial
cells with H. pylori leads to iNOS activation, formation of ONOO3 and is associated with an increase in
DNA fragmentation and apoptosis [114]. Protection
against ONOO3 mediated cell death may be aorded
by antioxidants such as ascorbic acid. For example
ascorbate prevented ONOO3 -induced apoptosis in
human intestinal epithelial cells either when added
before or concurrently with ONOO3 [115]. Various
models of insulin-dependent diabetes mellitus have
shown that the loss of L islet cells occurs by apoptosis mediated by ONOO3 formed by inltrating immune cells [116^123]. The level of the cellular antioxidant glutathione seems to be a critical factor
in neuronal susceptibility to peroxynitrite-induced
apoptosis, with high levels of glutathione being protective [124].
In certain circumstances the production of
ONOO3 may in fact be benecial, for instance, in-

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R.P. Patel et al. / Biochimica et Biophysica Acta 1411 (1999) 385^400

duction of ONOO3 formation in tumour cells can

lead to elimination of the disease. The anti-tumour
agent avone acetic acid has been shown to stimulate
ONOO3 production in subcutaneous mouse tumours
via the induction of iNOS. This ONOO3 production
correlated with the presence of apoptosis in the tumour and led to its dissolution [125].
8. Reactions of RNS to yield NO donors
From Fig. 1 it might be anticipated that RNS,
through a combination of oxidative and nitrosative
stress, would inhibit normal NO-dependent functions. However, in the intensive investigations of
the eects of ONOO3 in biological systems it was
demonstrated that ONOO3 possesses physiological
properties, which are similar to those of NO
[17,18,126^132]. These include the induction of blood
vessel relaxation and the inhibition of platelet aggregation and leucocyte adhesion to the endothelium
[17,133].
These NO-`like' eects of ONOO3 occur through
an NO-dependent mechanism since scavengers of
NO or inhibitors of guanylate cyclase abolish this
response in both platelets and artery ring segments
[128]. In vivo, systemic administration of ONOO3
transiently reduces mean arterial pressure in rats consistent with NO release [134]. With repeated administration, however, tachyphylaxis rapidly ensued, and
the blood pressure lowering eect of ONOO3 was
lost. This eect was also found on exposure of isolated perfused hearts to ONOO3 , and is similar to
the development of nitrate `tolerance' after chronic
usage of organic nitrates such as nitroglycerin
[126,136].
There are two potential sources for the formation
of compounds with NO-donating properties. The
rst of these is the reaction of ONOO3 with polyhydroxylated compounds such as glucose and glycerol, resulting in the incorporation of NO-containing
groups in these molecules [127,137]. The second is
the reaction with either low molecular weight or protein bound thiols [17,18,59]. Either of these reaction
products are moderately stable species, capable of
releasing NO on contact with tissues, resulting in
NO-dependent vasodilatation and inhibition of platelet aggregation [17,133]. Peroxynitrite can also in-

Fig. 4. Mechanisms underlying the vasorelaxant eect of peroxynitrite. Under conditions of vascular stress such as hypercholesterolaemia, NO is prevented from direct activation of guanylate cyclase (GC) due to its reaction with O3
2 . However,
ONOO3 can modify thiol-containing proteins (RSH) or polyhydroxylated compounds such as glucose (ROH), resulting in the
formation of NO donors. The reaction product of glucose and
ONOO3 is structurally and biochemically related to the organic
nitrites (RONO). RONO may be further modied to form an
S-nitrosothiol (RSNO) which is metabolised intracellularly to
release free NO, resulting in activation of GC [135]. Elevations
in cGMP are associated with a reduction in intracellular calcium and vessel relaxation. Peroxynitrite may also induce relaxation via the activation of ATP-sensitive potassium channels in
the VSMC membrane. Potassium eux through this channel is
associated with membrane hyperpolarisation and a decrease in
vascular excitability.

duce the formation of NO donors via reactions with

membrane impermeant sugars such as mannitol
[17,132]. The vasorelaxant activity associated with
these compounds may be due, in part, to the spontaneous release of low quantities of NO or the reductive metabolism at the cell surface.
Detailed structural analysis of the reaction products of glycerol and ONOO3 revealed the formation
of both glyceryl mononitrate and mononitrite, metabolites of nitroglycerin [137]. These products are
formed at sucient concentrations to exert biological
eects such as vessel relaxation. Treatment of arterial
tissues with diethyl maleate, an agent that depletes
tissue thiol groups, also blunts the relaxant eects of
ONOO3 . This result was interpreted as evidence for
ONOO3 -dependent nitrosation of cellular sulphydryl
groups, yielding a nitrosothiol compound, which can
be further metabolised to release free NO [128].
However, it is also consistent with the formation of
an S-nitrosothiol from an organic nitrite derived
from the reaction between ONOO3 and glucose
(Fig. 4). The vasorelaxant responses to ONOO3

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R.P. Patel et al. / Biochimica et Biophysica Acta 1411 (1999) 385^400

were similar in both intact and endothelium-denuded

canine coronary arteries, suggesting a direct action of
an NO-donating metabolite at the level of the vascular smooth muscle cell (VSMC) [129]. It is clear
that the nitrosative reactions resulting in the formation of NO donors may occur with a high chemical
yield in biological systems where NO and O3
2 are
generated simultaneously [59]. This is evident in the
treatment of platelets in which the inhibition of platelet aggregation by ONOO3 occurs before oxidation
reactions can be detected [17,133]. This no doubt
arises because of the exquisite sensitivity of biological
systems to NO-dependent eects. This may be particularly important in an inammatory setting where
it is likely that NO is inactivated by O3
2 . The NO
donors react much less readily with O3
2 than NO
thus preserving NO-dependent signalling pathways
under conditions where they would otherwise be inactive.
It is critical that appropriate control experiments
are performed when studying the vascular eects of
ONOO-derived NO donors in vitro. This is underscored by ndings of a recent study which suggest
that ONOO3 readily reacts with HEPES, a common
component of buers used in vessel reactivity studies,
to form an NO donating metabolite [138]. Topical
application of ONOO3 relaxes cerebral vessels by a
mechanism that is insensitive to blockade by a guanylate cyclase inhibitor [139]. This response was prevented by glyburide, a specic blocker of the ATPsensitive potassium channel (IKATP ). It was suggested that ONOO3 directly activates IKATP in vascular smooth muscle cells of cerebral arteries, resulting in membrane hyperpolarisation and vessel
relaxation [139].
Taken together these data suggest that, while oxidation is likely the primary reaction mechanism of
ONOO3 , nitration and nitrosation of proteins, carbohydrates and thiols result in the formation of compounds which act as NO donors. Reaction pathways
involved in NO donor formation are summarised in
Fig. 4.
9. Summary
It is well recognised that NO has an important role
in many physiological processes and we propose that

395

this be extended to include the generation of a range

of RNS. These compounds have two eects in biological systems. The rst of these is to eectively
remove NO-dependent responses, a fact that has
been well appreciated in our understanding of vascular disease such as atherosclerosis. The second is to
generate novel compounds which have distinct signalling properties which may either duplicate those
of NO or antagonise them. Although this makes the
investigation of the eects of RNS in biological systems yet more complex, it does emphasise the need to
complement a biochemical approach with experiments in which functional parameters are also measured.
Acknowledgements
Supported by National Institutes of Health Grants
Nos. HL48676, HL59685, HL51245, HL58115,
HL53601 and American Heart Association Grant
No. AL-G-960035.

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