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Home , Hyponitrite, Nitrite, Nitrosonium, Nitroxyl, Peroxynitrous acid

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Biochimica et Biophysica Acta 1411 (1999) 263^272

Review Relationships between nitric , , cation and

Martin N. Hughes *

Department of , King's College London, Strand, London WC2R 2LS, UK

Received 14 August 1998; received in revised form 5 November 1998; accepted 16 December 1998

Abstract

This review is concerned mainly with the three -related, but chemically distinct, species NO3,NOc and NO‡, with greatest emphasis being placed on the chemistry and biology of the nitroxyl ion. Biochemical routes for the formation of nitroxyl ion and methods for showing the intermediacy of this species are discussed, together with chemical methods for generating nitroxyl ion in solution. Reactions of nitroxyl ion with NOc, , iron centres in haem and with dioxygen are reviewed The significance of the reaction between NO3 and dioxygen as a source of peroxynitrite is assessed, and attention drawn to the possible significance of the spin state of the nitroxyl ion in this context. The biological significance of and the importance of S-nitrosothiols and certain metal nitrosyl complexes as carriers of NO‡ at physiological pH is stressed. Some features in the chemistry of peroxynitrite are noted. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: ; Nitroxyl anion; Peroxynitrite

Contents

1. Introduction ...... 264

2. The nitroxyl anion, NO3 ...... 264

3. Biochemical pathways for the production of nitroxyl ion ...... 265

4. Reactions of nitroxyl ion ...... 265 4.1. Reaction with metal centres to give nitrosyl complexes ...... 266 4.2. Reaction with thiols ...... 266 4.3. Reaction with the ...... 266 4.4. Cytotoxicity of nitroxyl ion ...... 267

5. Chemical routes for generating nitroxyl ion ...... 267

6. Photochemical decomposition of trioxodinitrate ...... 268

7. Electronic states of the nitroxyl ion ...... 268

* Fax: +44 (171) 873-2810; E-mail: [email protected]

0005-2728 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S0005-2728(99)00019-5

BBABIO 44732 23-4-99 264 M.N. Hughes / Biochimica et Biophysica Acta 1411 (1999) 263^272

8. The nitrosonium cation, NO‡ ...... 269

9. Peroxynitrite, ONOO3 ...... 269

10. Conclusions ...... 270

References ...... 270

1. Introduction under these conditions, the nitroxyl ion then reacts 3 3 with NO to give (NO)2 and (NO)3 [4^6]. Nitric This review deals with the relationships between oxide is also reduced to NO3 by Fe(II) in acidic the nitroxyl anion (NO3, oxonitrate(13)), nitric ox- solution, while nitroxyl ion can be oxidised to NO c 33 ide (NO , monoxide), the nitrosonium cati- by ferricyanide ion, [Fe(CN)6] . on (NO‡, nitrosyl cation) and peroxynitrite Interconversion of NO3,NOc and NO‡ can take (ONOO3, oxoperoxonitrate(13)), in which the nitro- place under cellular conditions, so all three species gen atom has formal positive oxidation states of I, II, must be considered in order to account fully for the III and III, respectively. The immense importance of biological activity of nitric oxide. The diversity and nitric oxide in the sciences and medicine needs no complexity of such activity is consistent with the view elaboration. Peroxynitrite has also attracted consid- that various species derived from NO play important erable attention as a species through which the cyto- biological roles [7]. Indeed, NO3,NOc and NO‡ toxic e¡ects of nitric oxide may be manifested under each exert unique toxic e¡ects on sporo- oxidising conditions, as peroxynitrite can be formed genes [8]. by reaction between nitric oxide and ion (Eq. 1) [1]. It should be noted, however, that Fukuto and Ignarro have recently suggested that the evi- 2. The nitroxyl anion, NO3 dence supporting the presence of peroxynitrite in bio- logical systems is indirect and potentially ambiguous This review will focus on the nitroxyl ion as the and that other mechanisms may explain NO-medi- importance of this species has been overlooked or ated toxicity under oxidative conditions in vivo [2]. rejected in the past. However, it must now be ac- The species nitroxyl ion and the nitrosonium cat- cepted that nitroxyl ion can be formed in the cellular ion are related to nitric oxide by reduction and oxi- milieu by several routes and that it can exert direct dation of NOc respectively, and each has a distinctive and varied e¡ects on biological and sys- chemistry unique to itself [3]. The fundamental chem- tems. Thus, it has been proposed that nitroxyl ion istry of these three species is best rationalised in may carry out reactions typical of the endothelial- terms of MO theory. The nitroxyl anion, isoelec- derived relaxing factor (EDRF), although the possi- tronic with dioxygen, has a bond order of 2. Nitric bility that nitroxyl ion is oxidised to NO before these oxide is a radical, with a bond order of 2.5, the un- reactions occur cannot be excluded. Claims have paired electron occupying an antibonding Z orbital. been made that nitroxyl ion is formed directly by Nitric oxide does not show a strong tendency to (NOS). Furthermore, its chem- dimerise. Loss of the unpaired electron gives NO‡, istry and biology are linked to that of peroxynitrite which has a bond order of 3 and is isoelectronic with as the nitroxyl ion reacts with dioxygen to give per- N2 and CO. Nitric oxide is di¤cult to oxidise, with a oxynitrite (Eq. 2). This reaction occurs in the autox- of 1.2 V. The electron a¤nity of idation of hydroxylamine in alkaline solution [9], one NOc will depend upon whether the NO3 product is route for the preparation of peroxynitrite. Several in the singlet or triplet state, but the reduction of reports have shown that co-ordination of NO to an nitric oxide to the nitroxyl ion can be carried out Fe(II) haem protein leads to reduction to give an by biological reducing agents. Nitroxyl ion is formed NO3 nitrosylhaem group which may then be fol- by reaction of NOc with hydrated electrons, although lowed by release of the nitroxyl ion. This may result

BBABIO 44732 23-4-99 M.N. Hughes / Biochimica et Biophysica Acta 1411 (1999) 263^272 265 in the formation of peroxynitrite under aerobic envi- ping of nitroxyl ion by thiols has been reported ear- ronments and so this pathway must also be consid- lier by Doyle et al. [20] and Turk and Hollocher [13]. ered in any assessment of the likelihood and extent RSH of intracellular formation of peroxynitrite. The par- RSH ‡ HNO ! RSNHOH!RSSR ‡ NH2OH ent acid of the nitroxyl ion (HNO) has pKa = 4.7 and 3† so is fully deprotonated at biological pH values

3 3 2HNO ! N2O ‡ H2O 4† NO ‡ O2 ˆ ONOO 1†

3 3 This reaction is important as it extends the modes NO ‡ O2 ˆ ONOO 2† of reactivity of S-nitrosothiols. These compounds Methods for the chemical generation of nitroxyl undergo homolytic cleavage to give NOc (and are ion in solution have recently been described [10]. often used as NOc releasers) although in practice they are usually more reactive as nitrosating agents (that is, they donate the NO‡ group to an nucleo- 3. Biochemical pathways for the production of phile of appropriate reactivity). The possibility that nitroxyl ion S-nitrosothiols can also donate nitroxyl ion to thiols emphasises further the intracellular signi¢cance of It was shown some time ago by Murphy and Seiss the species formed by oxidation or reduction of that superoxide dismutase catalyses the reversible re- NOc and has consequences in terms of redox balance duction of nitric oxide to nitroxyl ion [11]. The for- that may be signi¢cant in regulatory and other proc- mation of nitroxyl ion can be con¢rmed by trapping esses. Furthermore, the hydroxylamine-producing re- with metmyoglobin (Fe(III)) [12]. action leads to the formation of S^S bonds and so As noted above, the binding of nitric oxide to could simultaneously induce conformational changes iron(II) in a haem centre may lead to the release of in or receptor sites. nitroxyl anion, which in the absence of other reac- tions will dimerise to give . Examples include the nitric oxide reductase of Paracoccus de- 4. Reactions of nitroxyl ion nitri¢cans [13], cytochrome c [14] and haemoglobin [15]. In the case of cytochrome c, the released nitrox- The nitroxyl ion is a short-lived species in solution, yl ion reacted with dioxygen to give peroxynitrite, decomposing via dimerisation and dehydration to shown by the oxidation of dihydrorhodamine 123 give nitrous oxide (Eq. 4). The second-order rate [14]. In contrast to these examples of reductive for- constant for the dimerisation of HNO is 1.8U109 mation of nitroxyl ion, the oxidation of ion by dm3 mol31 s31 [4]. The rate of dimerisation will lignin peroxidase also leads to the release of nitroxyl vary considerably with concentration in view of the ion from the iron centre in the [16]. square dependence on [NO3] in the rate law. At Nitroxyl ion may also be formed by the oxidative neutral pH values, the life time of transient concen- decomposition of N-hydroxyarginine, the intermedi- trations of nitroxyl ion is likely to be milliseconds. ate formed during the NOS-catalysed oxidation of Nitroxyl ion reacts with a variety of targets. Some by oxygen [17]. There are also claims that of these reactions may be useful for establishing the NO3 is released under some conditions during the presence of nitroxyl ion as a reaction intermediate. NOS-catalysed oxidation of L-arginine [18], but these The reaction with thiols is a good example of such results may need further consideration. reactions and is discussed later. Alternatively, the Arnelle and Stamler have shown that the decom- nitroxyl ion dimerisation reaction may be exploited position of S-nitrosothiols in the presence of thiols to show conclusively the formation of NO3 by al- leads to the formation of hydroxylamine and nitrous lowing a reaction thought to produce NO3 to take oxide, and have interpreted these results in terms of a place in the presence of 15N sodium trioxodinitrate 15 23 15 3 reaction between nitroxyl ion (donated by the S-ni- (O NNNO2 ) which releases NO on decompo- trosothiol) and thiols (Eqs. 3 and 4) [19]. The trap- sition. If nitrous oxide with mass number 45 is

BBABIO 44732 23-4-99 266 M.N. Hughes / Biochimica et Biophysica Acta 1411 (1999) 263^272 formed, then cross dimerisation must have occurred a free group (unlike horse heart cytochrome between the 15NO3 released from the 15N-labelled c), and observing the formation of dimers cross- 14 3 trioxodinitrate and NO produced in the reaction linked via the L-cysteine residues. under study. Unlabelled trioxodinitrate may be used if the NO3 source molecule is 15N-labelled at the 4.3. Reaction with the oxygen molecule appropriate point. Another approach to testing for the intermediacy of the nitroxyl ion exploits the fact The reaction between nitroxyl ion and dioxygen to that the nitroxyl anion reacts rapidly with NO in a give peroxynitrite is of particular signi¢cance. Can reaction that is close to di¡usion controlled [4^6]. this be a stoichiometrically signi¢cant route for the The addition of an NO releaser to a reaction should production of peroxynitrite compared to the path- therefore inhibit any reaction attributable to nitroxyl way involving nitric oxide and superoxide? Second- anion. In these approaches, of course, complications order rate constants are 5.7U107 dm3 mol31 s31 and may arise from the addition of the or the NO3 1.9U1010 dm3 mol31 s31, respectively [23], favouring and NO releasers. the superoxide^nitric oxide pathway by a factor of 300. However, intracellular concentrations of super- 4.1. Reaction with metal centres to give nitrosyl oxide are very low, due to the e¤cient catalysis of its complexes by superoxide dismutase. Basal release of superoxide from cultured endothelial cells Nitroxyl ion formed during autoxidation of hy- is too low to be measured [24]. Concentrations of droxylamine at high pH reacts with oxygen to give dioxygen could be up to a thousand times greater the peroxynitrite anion [9]. The intermediacy of the than those of superoxide, supporting the suggestion nitroxyl ion in this process was con¢rmed by trap- that the reaction between dioxygen and nitroxyl ion 23 ping with the anion [Ni(CN)4] to give the violet is a signi¢cant peroxynitrite source. However, assess- tricyanonitrosylnickelate(II) anion, in which the ni- ment of the rate of reaction between nitroxyl ion and trosyl group is present as NO3 [9]. Nitroxyl ion dioxygen is complicated by two factors. The concen- formed by decomposition of the hydrogentrioxodini- tration of the nitroxyl ion is di¤cult to estimate 3 trate ion, HN2O3 , has been trapped using methae- (there are competing reactions, for example with moglobin or metmyoglobin [12]. The nitrosyl prod- haem and iron^sulfur centres and dimerisation). uct is EPR-detectable. Cytochrome d is nitrosylated The second factor involves the spin state of the ni- by the nitroxyl ion released from trioxodinitrate [21]. troxyl ion, which, like the isoelectronic dioxygen, can The nitroxyl ion, released from trioxodinitrate, has a exist in singlet or triplet states, with zero or two speci¢c inhibitory e¡ect on the production of hydro- unpaired electrons respectively. However, while di- gen via the phosphoroclastic pathway in C. sporo- oxygen has a triplet ground state, NO3 has a singlet genes: this has been attributed to the binding of ground state. Nitroxyl ion in the singlet state does the nitroxyl ion at an iron^sulfur centre [8]. not react with dioxygen. The thermal decomposition of trioxodinitrate (known to give singlet nitroxyl ion) 4.2. Reaction with thiols in oxygenated aqueous solution in the pH range 7^8 and at concentrations around 7U1035 mol dm33 The reaction with cysteine (Eq. 3) has been used to does not lead to the formation of , which establish the formation of nitroxyl ion. High concen- should be present if peroxynitrite had been formed trations of cysteine cause complete inhibition of the at these concentrations (M.N. Hughes, Bonner F.T. vasorelaxant activity of the trioxodinitrate group and M. Sherry, unpublished data). It appears, there- [22], con¢rming the involvement of nitroxyl ion, fore, that dioxygen reacts with the excited, triplet although this does not eliminate the possibility that state of the nitroxyl ion. Thus, the magnitude of the nitroxyl ion serves as a source of nitric oxide via the pathway for peroxynitrite formation involving oxidation. Sharpe and Cooper [14] have con¢rmed nitroxyl ion is di¤cult to place on a quantitative that ferrocytochrome c reduces nitric oxide to NO3 basis, especially as the electronic state of the nitroxyl by using the yeast ferrocytochrome c, which contains ion produced by particular pathways may not be

BBABIO 44732 23-4-99 M.N. Hughes / Biochimica et Biophysica Acta 1411 (1999) 263^272 267 known. Nevertheless, Sharpe and Cooper [14] have may be obtained by drying at 100³C. produced evidence to show that the nitroxyl anion NH OH ‡ RONO ‡ 2NaOC H ! formed by reduction of NOc by ferrocytochrome c 2 2 2 5 reacts with dioxygen to form peroxynitrite. These ROH ‡ Na2N2O3 ‡ 2C2H5OH 5† results show that peroxynitrite can be formed in the absence of superoxide by the reaction between The stoichiometry of the decomposition of trioxo- dioxygen and nitroxyl ion, a conclusion that empha- dinitrate is pH-dependent [29]. At pH values below 4, sises the potential signi¢cance of peroxynitrite as a the only product is nitric oxide. This reaction is ini- cytotoxic agent. tiated by and its onset at around pH 4 prob- ably re£ects the protonation of nitrite to give nitrous 4.4. Cytotoxicity of nitroxyl ion acid, followed by nitrosation of the hydrogentrioxo- 3 dinitrate anion HN2O3 . At pH values above 4, the Nitroxyl ion may exert toxic e¡ects apart from products are nitrous oxide (via dimerisation of ni- those arising from the formation of peroxynitrite. It troxyl ion) and nitrite. The formation of nitrite inhibits production via the phosphoroclas- alongside nitroxyl ion means that control experi- tic pathway in C. sporogenes by binding to an iron^ ments must be carried out with nitrite to con¢rm sulfur cluster, as shown by EPR [8]. that any e¡ects observed on a biological process Wink et al. [25] have shown that sodium trioxodini- are not due to nitrite ion. If 15N hydroxylamine is trate is toxic to Chinese hamster V79 lung ¢broblast used to synthesise sodium trioxodinitrate(23), then cells at concentrations of 2^4 mM. The presence of 15N nitroxyl ion will be produced on decomposition. ferricyanide in equimolar amounts protected against The ¢rst order rate constant for the decomposition these e¡ects by oxidising the nitroxyl ion to NO of trioxodinitrate is independent of pH between pH [25,26]. The toxicity of the trioxodinitrate was en- 4.4 and 8.1 and its value at 25³C is 6.75U1034 s31, hanced under aerobic conditions. Exposure of the with a half life of about 17 min. At 37³C, these cells to trioxodinitrate resulted in double strand values are 4.1U1033 s31 and 2.8 min, respectively 3 breaks in DNA. Peroxynitrite does not induce this [29,30]. The pKa values for H2N2O3 and HN2O3 type of cellular damage and so these e¡ects are as- are 2.39 and 9.36, respectively. Thus the reacting 3 sumed to be brought about by the nitroxyl anion. species is HN2O3 . Trioxodinitrate under these con- ditions of pH is stabilised by added nitrite, a product of the reaction, showing that an equilibrium exists 3 5. Chemical routes for generating nitroxyl ion between HN2O3 and the immediate products of ni- trogen^nitrogen bond cleavage (Eq. 6) [31]. Nitroxyl ion is usually generated in solution by the decomposition of either Angeli's , Na2N2O3 (via 3 6† the hydrogentrioxodinitrate(13) ion, HN2O3 ) or Pi- loty's acid, N-hydroxybenzenesulfonamide (which is commercially available). It should be noted that the salt `NaNO', prepared by the reaction of nitric oxide This reaction cannot involve cleavage of the nitro- with sodium in liquid , is dimeric and does gen^nitrogen double bond in trioxodinitrate, as this not act as a source of NO3 in solution. The rate of would give products in the triplet electronic state. reactions involving the nitroxyl ion will normally be The reverse reaction involves nitrite ion in the determined by the rate of decomposition of the ni- ground state and therefore singlet nitroxyl ion. By troxyl ion generating species. The concentrations of the principle of microscopic reversibility, the forward nitroxyl ion present in solution will be uncertain. reaction must therefore also involve singlet nitroxyl The synthesis of sodium trioxodinitrate, ion. Therefore, the decomposition of the hydrogen- Na2N2O3WH2O, is shown in Eq. 5 [27,28]. A variety trioxodinitrate ion involves cleavage of an nitrogen^ of organic have been used, including ethyl, nitrogen single bond, to give singlet nitroxyl ion. The isopropyl and butyl nitrates. The anhydrous form rate-determining step has accordingly been suggested

BBABIO 44732 23-4-99 268 M.N. Hughes / Biochimica et Biophysica Acta 1411 (1999) 263^272

[31] to involve tautomerisation of the species with a 6. Photochemical decomposition of trioxodinitrate nitrogen^nitrogen double bond into one with a nitro- gen^nitrogen single bond, which then undergoes N^ As noted above, self-decomposition of trioxodini- N bond cleavage. trate to give nitrite and nitroxyl ion in the presence Piloty's acid brings about , which has of dioxygen does not lead to the formation of per- been attributed to the release of the nitroxyl ion. oxynitrite. In contrast, the photochemical decompo- However, the decomposition of this compound to sition (using the mercury line at 253.7 nm) of the release NO3 in aqueous solutions is extremely slow trioxodinitrate anion in the presence of dioxygen at neutral pH (t1=2 about 80 h, at 25³C) and studies does give peroxynitrite [33], as shown by the forma- must be carried out under anaerobic conditions to tion and subsequent decay of a species with maxi- prevent rapid oxidative decomposition of Piloty's mum absorbance at 300 nm. Peroxynitrite was not acid [32]. It appears probable that the guanylyl cy- seen at pH 8, in accordance with its known instabil- clase-stimulating, vasodilator and anti-platelet activ- ity and probably the photolysis of ity of Piloty's acid are due to the nitric oxide released itself. The formation of peroxynitrite was observed at by this oxidative process [26]. The fact that cysteine higher pH values. In 0.1 mol dm33 NaOH solution, has no e¡ect on the biological activity of Piloty's acid the conversion of trioxodinitrate to peroxynitrite was under these conditions con¢rms that this is not due stoichiometric. Nitrite was also formed. Peroxynitrite to the formation of nitroxyl ion [26]. was not formed when the photochemical decomposi- Values of the ¢rst-order rate constant for the de- tion was carried out using dinitrogen-saturated solu- composition of Piloty's acid to nitroxyl ion increase tions, the products being nitrite (on a 1:1 stoichiom- with pH, as the anion of Piloty's acid is the kineti- etry) and nitrous oxide. This suggests that the triplet cally active species. The pKa of Piloty's acid is 9.29, state nitroxyl ion formed in the photochemical de- so the values of k1 level o¡ by about pH 11, with a composition of trioxodinitrate is converted quantita- 34 31 maximum value of k1 = 4.22U10 s when Piloty's tively to peroxynitrite. acid is fully deprotonated (Eqs. 7^9). At pH values 7.40 and 8.00 at 25³C, the half for the decom- position of Piloty's acid are 35 and 6 h, respectively 7. Electronic states of the nitroxyl ion [30]. The nitroxyl ion is isoelectronic with the oxygen C H SO NHOH ‡ OH3 ˆ C H SO NHO3 ‡ H O 6 5 2 6 5 2 2 molecule, which exists in a triplet ground state with 7† two unpaired electrons in the degenerate Z antibond- ing orbitals. In the singlet excited state, the electrons C H SO NHO3 ˆ HNO ‡ C H SO3 8† 6 5 2 6 5 2 are paired to give a diamagnetic molecule. In the gas 3 HNO ˆ H‡ ‡ NO3 9† phase, NO has a triplet ground state, while the singlet state is 72 kJ mol31 higher in energy [34]. Addition of the product benzenesul¢nate anion re- However, as argued above, the nitroxyl ion formed sults in decreased rates of decomposition of Piloty's on thermal decomposition in aqueous solution of the acid, suggesting reversibility of the decomposition of hydrogentrioxodinitrate anion is in the singlet, the anion (Eq. 8). This con¢rms that the other de- ground electronic state, and does not react with mo- composition product must be nitroxyl ion and sug- lecular oxygen. The nitroxyl anion formed on photo- gests that the singlet state anion is released from chemical decomposition in aqueous solution of triox- Piloty's acid, as well as from trioxodinitrate. Some odinitrate anion is in the triplet state and reacts with inhibitory e¡ects of Piloty's acid on the growth of molecular oxygen to give peroxynitrite. The nitroxyl food spoilage and other bacteria under anaerobic ion is a heteronuclear species with the negative conditions in media around pH 8 have been attrib- charge localised on the nitrogen atom (the parent uted to the release of the nitroxyl ion (Hughes, Cam- acid is HNO). This must lead to su¤cient splitting mack, Khan and Torres Martinez, unpublished of the degeneracy of the Z antibonding orbitals to work). result in a singlet ground state for the nitroxyl ion.

BBABIO 44732 23-4-99 M.N. Hughes / Biochimica et Biophysica Acta 1411 (1999) 263^272 269

Stanbury [35] has calculated the reduction poten- tively, a radical substrate species might be formed tials for the NO/1NO3 and NO/3NO3 couples to be that will react with NO. 30.35 and 0.39 V, respectively. The latter value is Nitrosation is an important process in cell bio- consistent with the suggestion that NOc can be re- chemistry. Receptor sites in control and signalling duced to NO3 by the hexaammineruthenium(II) cat- pathways often contain thiol groups. Nitrosation of ion [36]. these thiol groups (RS3) to give S-nitrosothiols (RSNO) could lead to homolysis of the S^N bond with loss of nitric oxide, the formation of S^S bonds 8. The nitrosonium cation, NO+ and conformational changes with considerable e¡ects upon cells. It is also possible that a series of trans- This cation, the oxidised form of nitric oxide, is nitrosations could occur, with the NO‡ group being related to and is the key species in the transferred from one carrier to another, until a crit- process of nitrosation, in which the NO‡ group is ical nitrosation step takes place. Compounds that act transferred (usually from a carrier compound) to a as NO‡ donors are e¡ective triggers of apoptosis in nucleophilic centre, often to a sulfur or nitrogen lone Swiss 3T3 ¢broblasts [41] and neuronal PC12 cells pair of electrons [37,38]. The nitrosonium cation is [42]. rapidly hydrolysed in aqueous solution to give ni- trous acid (Eq. 10). The equilibrium constant for this reaction is 1036:5 at 25³C [38] and the life time 9. Peroxynitrite, ONOO3 of NO‡ in is about 3U10310 s. There is an extensive literature on peroxynitrite. NO‡ ‡ H O ˆ H‡ ‡ HNO 10† 2 2 Much of this deals with reactions of peroxynitrite The use of aqueous solutions of salts containing with biological targets, that is covered elsewhere in ‡ the nitrosonium cation (e.g. [NO ][BF4]or this issue. These reactions may involve nitration and/ ‡ 3 [NO ][PF6 ]) in the expectation that these solutions or hydroxylation, as in the nitration of tyrosine res- contain the nitrosonium cation is unfortunate. The idues in proteins. Peroxynitrous acid is a stronger NO‡ cation is only found in aqueous solution at very oxidising agent than either nitric oxide or superoxide, high acidity. At pH values of biological relevance, from which it may be formed, and readily oxidises nitrosation is most likely to occur through the action thiols, ascorbate and lipids. It can carry out one- of NO‡ carriers such as S-nitrosothiols or possibly electron oxidations and two electron oxidations (by N-. S-Nitrosoglutathione is an excellent oxygen atom transfer). nitrosating agent, for example, and can transfer the It is generally accepted that activated macrophages NO‡ group to a variety of in the proc- produce peroxynitrite via the formation of NO and 3 ess now commonly called transnitrosation [37]. Ni- reaction with O2 [43]. While peroxynitrite produc- trosyl complexes in which the nitrosyl group is for- tion by macrophages is bene¢cial, its formation else- mally NO‡, for example , where is harmful [23] causing oxidative damage to ‡ Na2[Fe(CN)5(NO )], may be very e¡ective nitrosat- biological tissues and giving rise to various patho- ing agents at pH 7, particularly towards sulfur genic conditions. The toxic e¡ects of peroxynitrite centres. Such NO‡ nitrosyl complexes may be to cells and mitochondria is, therefore, a topic of formed intracellularly by reaction of NO with Fe(III) interest [44]. Its short life time of about 1 s at phys- centres. iological pH values presents di¤culties in such stud- Nitrosation at pH values of 7 and above by NO ies, although this life time is su¤cient to allow per- and NO2 have been studied by Challis and his co- oxynitrite to di¡use to crucial targets. As noted workers [39]. Nitric oxide cannot in isolation act as a earlier, there is evidence to show that the alternative nitrosating agent. It will function in this capacity in route for peroxynitrite formation, involving the reac- the presence of dioxygen [40] or other oxidising tion between dioxygen and nitroxyl ion, does occur agents, such as metal salts, because then NO will [14]. be oxidised to a species containing NO‡. Alterna- Peroxynitrite reacts rapidly with to

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3 give an adduct of composition ONO2CO2 [45^48]. 10. Conclusions Carbonate concentrations in physiological £uids are about 25 mmol dm33, so the formation of this ad- This survey of the reactions of the three simple duct takes may place more rapidly than do other diatomic species NO3,NOc and NO‡ and the rela- reactions associated with peroxynitrite toxicity. If tionships between them has demonstrated the com- this is the case, then, depending on the toxicity of plexity of their chemistry and the remarkable diver- this adduct, carbonate may either enhance peroxyni- sity of their biological behaviour. All this re£ects the trite toxicity or protect against it. non-innocence of the nitric oxide molecule. This £ex- Peroxynitrous acid ONOOH may be regarded as a ibility in chemical behaviour is maintained in intra- carrier of NO‡, although there is only limited evi- cellular molecules or such as S-nitrosothiols and dence to suggest that it can react as a nitrosating iron nitrosyl complexes, which maintain the redox agent. The presence of this group has a major in£u- £exibility of the NO group, e¡ectively serve as a ence on the peroxynitrous acid molecule. The strong store of these species and allow essential features of inductive e¡ect of NO‡ causes a weakening of the their chemistry to be manifested at pH 7. The rich O^O bond compared to that in , chemistry of small nitrogen compounds continues to with bond strengths of 90 and 170 kJ mol31, respec- be demonstrated in the reactions of the peroxynitrite tively. It also results in peroxynitrous acid having group, for which a number of issues are still open to one of the lowest pKa values of any peroxo acid, debate. with peroxynitrous acid and hydrogen peroxide hav- ing pKa values of about 6.8 (see [49]) and 15.7, re- spectively. Peroxynitrous acid isomerises to nitrate at References physiological pH values, although nitrite may also formed extensively under some conditions, for exam- [1] N.V. Blough, O. Za¤riou, Reaction of superoxide with ni- ple, in the presence of trace metals. The peroxynitrite tric oxide to form peroxynitrite in alkaline, aqueous solution, anion is stable with respect to decomposition. The Inorg. Chem. 24 (1985) 3504^3509. question of whether the decomposition and reactions [2] J.M. Fukuto, L.J. Ignarro, In vivo aspects of nitric oxide of peroxynitrous acid involves homolysis of the O^O (NO) chemistry: does peroxynitrite (-OONO) play a major bond to give OH and NO radicals, which either may role in cytotoxicity?, Acc. Chem. Res. 30 (1997) 149^152. 2 [3] F.T. Bonner, G. Stedman, The chemistry of nitric oxide and recombine to give nitrate or react with substrates, redox-related species, in: M. Feelisch, J.S. Stamler (Eds.), has been the subject of debate for many years. Re- Methods in Nitric Oxide Research, Wiley, Chichester, cently, stopped £ow studies on the isomerisation of 1996, pp. 341^356. peroxynitrous acid at high pressure have shown that [4] M. Gratzel, S. Taniguchi, A. Henglein, Pulsradiolytische Un- the rate of isomerisation depends little on the pres- tersuchung kurzlebiger Zwischenprodukte der NO-Reduk- sure, with a volume of activation of 1.7 cm3 mol31 tion in wassriger Losung, Ber-Bunsen-Ges. Phys. Chem. 74 (1970) 1003. [49]. This is not consistent with a mechanism of [5] W.A. Seddon, M.J. Young, Pulse radiolysis of nitric oxide in isomerisation in which the rate-determining step is aqueous solution, Can. J. Chem. 48 (1970) 393^394. homolysis of the peroxide bond to give free hy- [6] W.A. Seddon, J.W. Fletcher, F.C. Sopchyshyn, Pulse radiol- droxyl and radicals. However, 15N ysis of nitric oxide in aqueous solution, Can. J. Chem. 51 CIDNP NMR studies on the nitration of tyrosine (1973) 1123^1130. [7] S.A. Lipton, Y.-B. Choi, Z.-H. Pan, S.Z. Lei, H.-S. Chen, suggest that this reaction occurs by a radical proc- N.J. Sucher, J. Loscalzo, D.J. Singel, J.S. Stamler, A redox- ess, implying that peroxynitrite does cleave homo- based mechanism for the neurodestructive e¡ects of nitric lytically [50]. Nevertheless, it has been pointed out oxide and related compounds, Nature 364 (1993) that the question of whether or not peroxynitrite 626^629. undergoes homolysis or reacts via a trans inter- [8] S. Maraj, S. Khan, X.-Y. Cui, R. Cammack, C.L. Joannou, mediate is not relevant to biology. Peroxynitrite M.N. Hughes, The interaction of NO and redox-related spe- cies with biological targets, Analyst 120 (1995) 699^703. formed in blood vessels will react with carbon diox- [9] M.N. Hughes, H.G. Nicklin, The autoxidation of hydroxyl- ide while peroxynitrite formed in cells will oxidise in alkaline solution, J. Chem. Soc. A, (1971) 164^171. thiols [51]. [10] Hughes, M.N., R. Cammack, Synthesis, chemistry and ap-

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