Formation of Nitrating and Chlorinating Species by Reaction of Nitrite with Hypochlorous Acid

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO−). We show here that reaction of nitrite (NO−2), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO−2 with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO−2/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO−2/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO−2/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO−2/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (·NO2) and tyrosyl radical. We propose that NO−2 reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO−. Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO−). We show here that reaction of nitrite (NO−2), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO−2 with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO−2/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO−2/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO−2/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO−2/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (·NO2) and tyrosyl radical. We propose that NO−2 reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO−. INTRODUCTIONNitrogen monoxide (nitric oxide, ·NO) 1The abbreviations used are: ·NOnitric oxideO2superoxideHOClhypochlorous acidNO−2nitriteONOO−peroxynitriteONOOHperoxynitrous acidNO−3nitrateNO2-Tyr3-nitrotyrosineCl-Tyr3-chlorotyrosineHPA4-hydroxyphenylacetic acidNO2-HPA3-nitro-4-hydroxyphenylacetic acidCl-HPA3-chloro-4-hydroxyphenylacetic acidCl-PhechlorophenylalanineNATN-acetyl-L-tyrosineNAPN-acetyl-L-phenylalanineMPA4-methoxyphenylacetic acidCl-NO2nitryl chlorideCl-ONOchlorine nitriteROSreactive oxygen speciesRNSreactive nitrogen speciesHPLChigh pressure liquid chromatographyPDAphotodiode array. is produced by a variety of cells through the activity of constitutive and inducible forms of nitric oxide synthase (1Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Google Scholar). ·NO is an important endogenous mediator in such diverse biochemical and physiological processes as neurotransmission, smooth muscle relaxation, platelet aggregation and adhesion, macrophage-mediated cytotoxicity, and learning and memory (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar, 3Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Google Scholar). Although basal levels of free ·NO are normally quite low (nanomolar), local ·NO concentrations have been shown to increase to levels ranging from 4 to 30 µM under pathologic conditions (4Hooper D.C. Ohnishi S.T. Kean R. Numagami Y. Dietzschold B. Koprowski H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5312-5316Google Scholar, 5Malinski T. Zhang Z.G. Chopp M. J. Cerebral Blood Flow Metab. 1993; 13: 355-358Google Scholar).·NO reacts at a near diffusion-controlled rate with superoxide (O2) (k = 6.7 × 109M−1 s−1) (6Huie R.E. Padmaja S. Free Rad. Res. Commun. 1993; 18: 195-199Google Scholar) to form the cytotoxic species peroxynitrite (ONOO−). The formation of ONOO− is thought to be responsible, at least in part, for the observed toxicity associated with ·NO (7Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Google Scholar, 8Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Google Scholar). At physiological pH the protonated form of ONOO−, peroxynitrous acid (ONOOH) (pKa = 6.8), is highly unstable and rapidly decomposes to nitrate (NO−3). ONOOH is thought to 1) react directly with biological molecules via a vibrationally excited intermediate (ONOOH*), 2) decompose by homolytic dissociation to form nitrogen dioxide (·NO2) and the hydroxyl radical (·OH), or 3) by heterolytic dissociation to form the nitryl cation (nitronium ion, NO+2) (reviewed in Ref. 9Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722Google Scholar). ONOO−/ONOOH reacts with proteins, leading to the oxidation of cysteine, methionine, and tryptophan residues, and can induce protein carbonyl formation and nonspecific fragmentation (10Ischiropoulos H. Al-Mehdi A.B. FEBS Lett. 1995; 364: 279-282Google Scholar, 11Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Google Scholar, 12Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar). In addition, ONOO−/ONOOH can react readily with phenolic compounds to form nitrated, hydroxylated, and dimerized products (13Halfpenny E. Robinson P.L. J. Chem. Soc. (Lond.). 1952; : 939-946Google Scholar, 14Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Google Scholar, 15Van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Google Scholar, 16Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Google Scholar, 17Van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Google Scholar), and nitration of free tyrosine, or tyrosine in proteins, has served as a “marker” and “index” of ONOO− formation in vivo.Based upon tyrosine nitration assays and the formation of “peroxynitrite-specific” luminescence, stimulated macrophages (18Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Google Scholar), neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar), and endothelial cells (20Kooy N.W. Royall J.A. Arch. Biochem. Biophys. 1994; 310: 352-359Google Scholar) have been proposed to form significant quantities of ONOO− in vitro. In fact, the detection of 3-nitrotyrosine (NO2-Tyr) in a variety of pathologic conditions in vivo, such as inflammatory lung disease (21Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Google Scholar), atherosclerosis (22Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biochem. Hoppe-Seyler. 1994; 375: 81-88Google Scholar), and rheumatoid arthritis (23Kaur H. Halliwell B. FEBS Lett. 1994; 350: 9-12Google Scholar), has been attributed to ONOO− formation. However, in all of these cases direct proof for the production of ONOO− in biological systems is lacking, even though its formation in vivo is favorably predicted (24Squadrito G.L. Pryor W.A. Chem. Biol. Interact. 1995; 96: 203-206Google Scholar).Under inflammatory conditions, multiple well characterized reactive oxygen species (ROS) are produced from phagocytic cells (25Miller R.A. Britigan B.E. J. Invest. Med. 1995; 43: 39-49Google Scholar). For instance, stimulated neutrophils and macrophages produce significant levels of superoxide (O2) and hydrogen peroxide (H2O2) as a result of the activation of the respiratory burst oxidase (26Winterbourn C.C. Das D.K. Essman W.B. Oxygen Radicals: Systemic Events and Disease Processes. Karger, Basel, Switzerland1990: 31Google Scholar). In the case of neutrophils, some of the H2O2 that is produced under these conditions is converted to the strong oxidant hypochlorous acid (HOCl) by the action of myeloperoxidase as shown in Reaction 1. H2O2+H++C1−→HOC1+H2O REACTION 1HOCl produced from activated human neutrophils has been shown to react with amines (taurine, lysine, and arginine) and tyrosine to form N-chloramines and 3-chlorotyrosine (Cl-Tyr), respectively (27Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Google Scholar, 28Weiss S.J. Lampert M.B. Test S.T. Science. 1983; 222: 625-628Google Scholar), where the latter has been proposed to serve as a selective marker of HOCl production in vivo (29Kettle A.J. FEBS Lett. 1996; 379: 103-106Google Scholar).In addition to ROS, macrophages (30Marletta M.A. Yoon P.S. Iyengar R. Leaf C.D. Wishnok J.S. Biochemistry. 1988; 27: 8706-8711Google Scholar) and neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar) can also simultaneously produce large fluxes of ·NO through the activation of inducible nitric oxide synthase; however, the ability of human neutrophils to produce ·NO is debated (31Miles A.M. Owens M.W. Milligan S. Johnson G.G. Fields J.Z. Ing T.S. Kottapalli V. Keshavarzian A. Grisham M.B. J. Leukocyte Biol. 1995; 58: 616-622Google Scholar). Once formed, ·NO can react with several biological targets, primarily thought to involve heme-iron, hyperreactive sulfhydryls and protein radicals (32Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar, 33Lepoivre M. Flaman J.-M. Bobé P. Lemaire G. Henry Y. J. Biol. Chem. 1994; 269: 21891-21897Google Scholar, 34Eiserich J.P. Butler J. van der Vliet A. Cross C.E. Halliwell B. Biochem. J. 1995; 310: 745-749Google Scholar). ·NO can also react with O2 in aqueous solution to produce nitrite (NO−2) via a complex mechanism thought to involve a variety of reactive nitrogen species (RNS) including ·NO2 and dinitrogen trioxide (N2O3) (35Ignarro L.J. Fukuto J.M. Griscavage J.M. Rogers N.E. Byrns R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8103-8107Google Scholar). In fact, NO−2 has been used as a marker of ·NO production in vitro and in vivo and has been shown to reach concentrations of up to 4 µM in synovial fluid from patients with rheumatoid arthritis (36Farrell A.J. Blake D.R. Palmer R.M.J. Moncada S. Ann. Rheum. Dis. 1992; 51: 1219-1222Google Scholar) and as high as 20 µM in human airway fluids (37Gaston B. Reilly J. Drazen J.M. Fackler J. Ramdev P. Arnelle D. Mullins M.E. Sugarbaker D.J. Chee C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10957-10961Google Scholar). These RNS produced during an inflammatory response could theoretically react with a number of ROS to form various novel species. Indeed, the interaction of HOCl with ·NO or NO−2 has been proposed to form species capable of nitrosylating and nitrating organic substrates (38Koppenol W.H. FEBS Lett. 1994; 347: 5-8Google Scholar, 39Kono Y. Biochem. Mol. Biol. Int. 1995; 36: 275-283Google Scholar).The present study was undertaken to examine the potential interactions of ·NO-derived RNS with the inflammatory oxidant HOCl in an attempt to characterize more fully the various species that may be formed under complex physiological inflammatory conditions. Our results indicate that NO−2 reacts with HOCl to form an intermediate species, postulated to be nitryl chloride (Cl-NO2) and/or chlorine nitrite (Cl-ONO), that is capable of nitrating, chlorinating, and dimerizing phenolic compounds including tyrosine. We propose that the formation of Cl-NO2 and/or Cl-ONO by this reaction represents a novel mechanism of inflammation-mediated biological damage, and offers an additional or alternative mechanism of tyrosine nitration independent of ONOO− formation.DISCUSSIONAlthough the mechanisms of biomolecular damage and pathology induced by individual inflammatory oxidants are in general well characterized, an understanding of the complex interactions of ROS and RNS that are likely to occur at sites of inflammation is only just beginning to emerge. The studies reported herein show that the interactions of RNS and HOCl may be important under inflammatory conditions in vivo. We have shown that NO−2, the autoxidation product of ·NO in biological fluids, reacts with HOCl to produce a species that can nitrate, chlorinate, and dimerize biologically relevant phenolic compounds such as tyrosine, both free and within protein. The detection of NO2-Tyr in a variety of pathologic states (21Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Google Scholar, 22Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biochem. Hoppe-Seyler. 1994; 375: 81-88Google Scholar, 23Kaur H. Halliwell B. FEBS Lett. 1994; 350: 9-12Google Scholar) has been used to indicate the formation of ONOO− in vivo. However, reaction of tyrosine with the products of the NO−2/HOCl reaction also forms NO2-Tyr. Hence, our results suggest that NO2-Tyr should not be regarded as a specific marker of ONOO− formation, but only as a marker of RNS.Mechanism of NO−2/HOCl ReactionIt has long been thought (48Anbar M. Taube H. J. Am. Chem. Soc. 1958; 80: 1073-1077Google Scholar) that the reaction of NO−2 with HOCl represented a classical example of an oxygen atom transfer reaction producing NO−3. NO2−+HOC1→HC1+NO3− REACTION 2However, this type of mechanism does not easily explain the nitration and chlorination reactions observed in our studies. Our data suggest a more complex mechanism involving the formation of reactive nitrating and chlorinating intermediates. One-electron oxidation of NO−2 by HOCl, producing the reactive radical species Cl· and ·NO2 is one possible pathway. Since HOCl is a poor one-electron oxidant, having an estimated one-electron reduction potential (E′0) in the range of +0.17 to +0.26 V at pH 7 (38Koppenol W.H. FEBS Lett. 1994; 347: 5-8Google Scholar), it is unlikely that a one-electron oxidation mechanism contributes, since the E value for the ·NO2/NO−2 couple is approximately +1.04 V (49Wardman P. J. Phys. Chem. Ref. Data. 1989; 18: 1637-1755Google Scholar). In contrast, HOCl is a strong two-electron oxidant (E′0 = +1.08 V) (38Koppenol W.H. FEBS Lett. 1994; 347: 5-8Google Scholar) and would favor the conversion of NO−2 to the nitryl cation (NO+2) or an “NO+2-like” species. In addition to a direct two-electron oxidation of NO−2 by HOCl, a bimolecular substitution reaction between these two reactants could be involved. In fact, contrary to the reaction mechanism previously reported (48Anbar M. Taube H. J. Am. Chem. Soc. 1958; 80: 1073-1077Google Scholar), Johnson and Margerum (45Johnson D.W. Margerum D.W. Inorg. Chem. 1991; 30: 4845-4851Google Scholar) have suggested that HOCl reacts with NO−2 by Cl+ transfer, rather than O atom transfer, to yield the intermediate Cl-NO2, which then hydrolyzes to NO−3.The absorbance spectrum of the product of the reaction between NO−2 and HOCl (Fig. 5B) was found to be similar to that of authentic Cl-NO2 (Fig. 5C). The spectrum of the product(s) of the NO−2/HOCl reaction is typical of alkyl nitrites (R-ONO) (50Haszeldine R.N. J. Chem. Soc. (Lond.). 1953; : 2525-2527Google Scholar) and therefore could also indicate the formation of a Cl-O-bonded species. In fact, the transfer of Cl+ to the negatively charged oxygen atom in NO−2 is likely and would produce the transient intermediate species Cl-ONO. It is possible that both reactions occur (Fig. 9), the extent to which each pathway initially predominates under neutral aqueous conditions is not known. Cl-ONO can exist as both the cis- and trans-rotamers (Fig. 9), where ab initio calculations predict that the energy difference between the two rotamers is approximately 3 kcal/mol, with the cis rotamer being the more stable (51Lee T.J. J. Phys. Chem. 1994; 98: 111-115Google Scholar). An analogy can be drawn between Cl-ONO and HO-ONO (peroxynitrous acid), where the energy difference between cis- and trans-HO-ONO is also calculated to be approximately 3 kcal/mol (52Tsai J.-H.M. Harrison J.G. Martin J.C. Hamilton T.P. van der Woerd M. Jablonsky M.J. Beckman J.S. J. Am. Chem. Soc. 1994; 116: 4115-4116Google Scholar). Once formed, Cl-ONO can readily isomerize to Cl-NO2 (53Tevault D.E. Smardzewski R.R. J. Chem. Phys. 1977; 67: 3777-3784Google Scholar). We propose that intermediate Cl-ONO can isomerize in aqueous solution to Cl-NO2 by at least two mechanisms (Fig. 9): 1) intramolecular rearrangement of trans-Cl-ONO involving migration of the chlorine atom to the nitrogen atom forming Cl-NO2, or 2) unimolecular homolysis of the Cl-O bond in Cl-ONO to form a geminate pair of solvent-caged radicals Cl· and ·NO2, which undergo cage return to either reform Cl-ONO or by recombination to form Cl-NO2 (Fig. 9). Some of the solvent-caged Cl· and ·NO2 can escape as “free” radicals and could potentially explain, in part, the radical mechanisms involved in the nitration reactions we observed in the NO−2/HOCl reaction. Since Cl-NO2 is predicted to be 10.7 and 13.8 kcal/mol lower in energy than cis- and trans-Cl-ONO (51Lee T.J. J. Phys. Chem. 1994; 98: 111-115Google Scholar), respectively, the isomerization of Cl-ONO to Cl-NO2 is a favorable process that shifts the equilibrium toward Cl-NO2. Isomerization of cis Cl-ONO to Cl-NO2 is probably not likely, because the large size of the chlorine atom, which would presumably preclude the migration of the chlorine atom to the nitrogen atom and, hence, the trans-rotamer of Cl-ONO, is probably the species that isomerizes to Cl-NO2, analogous to the decomposition of trans-peroxynitrous acid (trans-HO-ONO). Whereas the isomerization of trans-HO-ONO leads to nitric acid (HO-NO2), an unreactive end product, isomerization of Cl-ONO produces another highly reactive species (Cl-NO2). Hence, Cl-ONO and the product of isomerization, Cl-NO2, may both be reactive oxidants with nitrating and chlorinating activity.Decomposition Products of Cl-NO2 as Reactive IntermediatesWe have shown that the product(s) of the reaction between NO−2 and HOCl, authentic Cl-NO2, or the NO+2 species (NO2BF4) react with tyrosine to form NO2-Tyr and dityrosine. Although none of these reactants are themselves radicals, formation of dityrosine suggests the involvement of intermediate tyrosyl radicals. The nitration of aromatic compounds by NO+2 is often thought to be a classical electrophilic aromatic substitution reaction, but there is strong evidence implicating electron transfer reactions and radical intermediates in these pathways (54Perrin C.L. J. Am. Chem. Soc. 1977; 99: 5516-5518Google Scholar). This reaction mechanism involves electron transfer from the aromatic to NO+2, followed by radical pair collapse, and it would explain the detection of dityrosine in our studies. Hence, we are unable to distinguish between a nitration mechanism involving ·NO2 or NO+2 based solely on the formation of dityrosine. However, a divergence in the characteristics of the reaction mechanisms between NO+2 and the reactive nitrating species formed by the reaction of NO−2 with HOCl is evident in their reactions with MPA, the O-methylated derivative of HPA, a substrate incapable of forming phenoxyl radicals. Whereas NO+2 appears capable of nitrating MPA, both the product(s) of the NO−2/HOCl reaction and synthetic Cl-NO2 fail to do so. Similarly, the inability of NO−2/HOCl and Cl-NO2 to nitrate phenylalanine further argues against NO+2 as the species involved in tyrosine nitration.There is evidence suggesting that the reaction of Cl-NO2 with alkenes and aromatic compounds involves homolytic processes yielding free radical intermediates (42Shechter H. Conrad F. Daulton A.L. Kaplan R.B. J. Am. Chem. Soc. 1952; 74: 3052-3056Google Scholar), probably involving both Cl· and ·NO2. Collis et al. (43Collis M.J. Gintz F.P. Goddard D.R. Hebdon E.A. Minkoff G.J. J. Chem. Soc. (Lond.). 1958; : 445-451Google Scholar) have found that Cl-NO2 decomposes at room temperature by homolysis to form Cl2 and ·NO2 as shown in Reaction 3, whereby these spontaneous decomposition products may be responsible, at least in part, for the chlorinating and nitrating behavior of Cl-NO2 in our experiments. We suggest that phenolic nitration mediated by the NO−2/HOCl reaction involves ·NO2. 2Cl−NO2→(2Cl·→Cl2)+(2·NO2⇌N2O4) REACTION 3While the nitration reactions we observed appear to be radical-mediated, chlorination of aromatic amino acids such as phenylalanine (Fig. 8) appears to be executed largely by electrophilic aromatic substitution. In general, chlorination of aromatic compounds by HOCl, tert-butyl hypochlorite, and Cl2 has been shown to be mediated by an ionic rather than a free radical mechanism (47Watson W.D. J. Org. Chem. 1974; 39: 1160-1164Google Scholar). The nearly 2-fold increase in the relative formation of the m-Cl-Phe isomer by reactions of phenylalanine with both NO−2/HOCl and Cl-NO2 (Fig. 8), however, suggests the potential contribution of a less selective mechanism of chlorination, potentially involving Cl·. An active chlorinating species common to HOCl and Cl-NO2 appears to be Cl2. In fact, the formation of Cl2 from HOCl and Cl-NO2 can be rationalized and would explain the similarities in their chlorinating ability. HOCl is in equilibrium with Cl2 in aqueous solution as shown in Reaction 4. The formation of Cl2 from Cl-NO2 has been proposed to occur by 1) the homolysis of two molecules of Cl-NO2 to form two Cl· which combine to form Cl2 (Reaction 3), and 2) the reaction of Cl-NO2 with H2O (43Collis M.J. Gintz F.P. Goddard D.R. Hebdon E.A. Minkoff G.J. J. Chem. Soc. (Lond.). 1958; : 445-451Google Scholar) as shown in Reaction 5. HOC1+H++C1−⇄Cl2+H2O REACTION 4 3Cl−NO2+H2O→Cl2+NOCl+2NO3−+2H+ REACTION 5Although convincing evidence suggests an electrophilic substitution mechanism for these chlorination reactions, the possibility of a mechanism involving the addition of Cl· to the aromatic ring cannot be excluded for reactions involving Cl-NO2 or NO−2/HOCl.Direct Reactions of Cl-NO2/Cl-ONO with TyrosineThe mechanisms of chlorination and nitration discussed thus far have primarily involved species derived from the decomposition of either Cl-NO2 or Cl-ONO. However, as predicted by the stoichiometry of Reactions 3 and 5, these pathways are particularly favored when Cl-NO2 or Cl-ONO are present at high concentrations. In vivo, however, Cl-NO2 and Cl-ONO would be expected to be produced at rates that may favor the direct reaction of either species with biological substrates that are present in relative excess. In nonpolar organic solvents Cl-NO2 has been shown to be an efficient agent for the nitration of aromatic compounds of intermediate reactivity (55Price C.C. Sears C.A. J. Am. Chem. Soc. 1953; 75: 3276-3277Google Scholar). However, an increase either in the reactivity of the aromatic substrate (from benzene to phenol) or in the polarity of the solvent causes a marked decrease in the nitrating efficiency of Cl-NO2 and a concomitant increase in the yield of chlorinated products (56Gintz F.P. Goddard D.R. Collis M.J. J. Chem. Soc. (Lond.). 1958; : 445-451Google Scholar). In fact, Obermeyer et al. (57Obermeyer A. Borrmann H. Simon A. J. Am. Chem. Soc. 1995; 117: 7887-7890Google Scholar) argued against the localization of a positive charge on the “nitryl” group of Cl-NO2, where the structural characteristics of Cl-NO2 contrast those of typical stable nitryl salts (i.e., NO+2BF−4). Hence, reactions involving activated aromatic substrates such as tyrosine coupled with aqueous conditions would increase aromatic chlorination by Cl-NO2, suggesting a change from Cl−NO+2 character to a species with considerable Cl+NO−2 character. Our data suggest that Cl-NO2 has significant Cl+ character in aqueous solution, and it is this functionality of Cl-NO2 that dictates its reactivity.We propose that Cl+NO−2 can react directly with tyrosine via electron transfer to yield an intermediate radical pair (tyrosyl radical-Cl·-NO−2) (Fig. 10). Radical pair collapse of this complex leads to the rapid formation of Cl-Tyr and NO−2 (Fig. 10, reaction A), and is the major product formed by this reaction. This electron transfer-mediated reaction mechanism is analogous to the nitration of phenolic substrates by NO+2 (54Perrin C.L. J. Am. Chem. Soc. 1977; 99: 5516-5518Google Scholar). Dissociation of the radical pair complex and subsequent oxidation of NO−2 by Cl· (a strongly oxidizing species, E′0 of Cl·/Cl− = +2.2-2.6 V (Ref. 49Wardman P. J. Phys. Chem. Ref. Data. 1989; 18: 1637-1755Google Scholar)) results in the formation of “free” tyrosyl radical and ·NO2 (Fig. 10, reaction B). Tyrosyl radical and ·NO2 can rapidly combine to yield NO2-Tyr (k = 3 × 109M−1 s−1 (Ref. 44Prütz W.A. Mönig H. Butler J. Land E.J. Arch. Biochem. Biophys. 1985; 243: 125-134Google Scholar)), and dityrosine formation can be envisaged by the combination of two tyrosyl radicals (Fig. 10, reactions C and D). This proposed mechanism predicts that the yields of the various tyrosine modification products will be on the order of Cl-Tyr > NO2-Tyr > dityrosine, consistent with the data presented herein. The proposed reaction pathway also illustrates the dependence of radical intermediates in the nitration of phenolic compounds by Cl-NO2, as suggested by our data.Fig. 10Proposed mechanisms for the direct reactions of tyrosine with Cl-NO2. The direct reaction of Cl-NO2 with tyrosine proceeds by electron transfer from tyrosine to Cl+NO−2, resulting in an intermediate radical pair (tyrosyl radical-Cl·). Radical pair collapse leads to the formation of Cl-Tyr and NO−2 (A) as major products. Dissociation of the complex from the solvent cage allows Cl· to oxidize NO−2 to ·NO2 (B), which can combine with simultaneously formed “free” tyrosyl radical to yield NO2-Tyr (C). Dityrosine formation can be envisaged by the combination of two tyrosyl radicals (D).View Large Image Figure ViewerDownload (PPT)Since Cl-ONO is a potential transient intermediate in the formation of the reactive species Cl-NO2 (Fig. 9), part of the reactivity of NO−2/HOCl may be attributed to Cl-ONO. Analogous to a proposed mechanism of ONOOH reactivity (9Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722Google Scholar, 58Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Google Scholar), a vibrationally excited intermediate derived from trans-Cl-ONO (Cl-ONO*) may be formed during its isomerization to Cl-NO2 and contribute to nitration and chlorination of tyrosine by direct reaction. The reaction mechanisms we propose for Cl-NO2 and Cl-ONO are analogous to those recently determined for ONOOH, whereby both direct and indirect reactions with oxidizable substrates can occur (59Goldstein S. Czapski G. Inorg. Chem. 1995; 34: 4041-4048Google Scholar). A more detailed examination of the reaction kinetics and thermodynamic considerations is necessary in order to elucidate which of the proposed mechanisms predominate.Physiological Relevance and Biological ImplicationsThe activation and accumulation of neutrophils at sites of tissue injury, leading to the formation of HOCl and other ROS/RNS, is an essential feature of inflammation. Our data suggest that the reaction of HOCl with NO−2, derived from ·NO produced by other phagocytes (30Marletta M.A. Yoon P.S. Iyengar R. Leaf C.D. Wishnok J.S. Biochemistry. 1988; 27: 8706-8711Google Scholar), endothelial cells (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar), or epithelial cells (37Gaston B. Reilly J. Drazen J.M. Fackler J. Ramdev P. Arnelle D. Mullins M.E. Sugarbaker D.J. Chee C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10957-10961Google Scholar), may be a contributing pathway operative in tissue injury at sites of inflammation. Moreover, since Cl-NO2 is conceivably formed in vivo and is capable of nitrating tyrosine residues, our findings may imply a role for this reaction pathway where NO2-Tyr is detected in cases of acute inflammatory lung injury (21Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Google Scholar), atherosclerosis (22Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biochem. Hoppe-Seyler. 1994; 375: 81-88Google Scholar), and rheumatoid arthritis (23Kaur H. Halliwell B. FEBS Lett. 1994; 350: 9-12Google Scholar), a phenomena previously ascribed to ONOO− formation. In fact, increased levels of NO−2 have been observed in similar cases (36Farrell A.J. Blake D.R. Palmer R.M.J. Moncada S. Ann. Rheum. Dis. 1992; 51: 1219-1222Google Scholar, 60Gaston B. Drazen J.M. Loscalzo J. Stamler J.S. Am. J. Respir. Crit. Care Med. 1994; 149: 538-551Google Scholar, 61Chester A.H. O'Neil G.S. Moncada S. Tadjkarimi S. Yacoub M.H. Lancet. 1990; 336: 897-900Abstract Full Text PDF Scopus (214) Google Scholar) and further suggest the potential involvement of this pathway in vivo. Recent studies have demonstrated that myeloperoxidase, the phagocytic enzyme that catalyzes HOCl formation, is a component of sputum from cystic fibrosis patients (62Mohammed J.R. Mohammed S.S. Pawluk L.J. Bucci D.M. Baker N.R. Davis W.B. J. Lab. Clin. Med. 1988; 112: 711-720Google Scholar), as well as other inflammatory lung diseases, and of human atherosclerotic tissue (63Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Google Scholar), underscoring the potential importance of HOCl in the pathology of each of these cases. We propose, then, that tyrosine nitration by Cl-ONO and/or Cl-NO2, formed by the reaction of NO−2 with HOCl, represents an important and additional mechanism for inflammation-mediated tyrosine nitration in vivo, independent of ONOO− formation.Our findings indicate that NO−2 may not be an appropriate marker of ·NO production by neutrophils or at sites of inflammation, since it is potentially removed by reaction with simultaneously produced HOCl. Determination of ·NO production in tissues and fluids of patients with acute and chronic inflammation, as measured by NO−2“accumulation,” is likely a gross underestimate. Moreover, NO−2 has been shown to modulate the bactericidal activity of HOCl, its mechanism proposedly mediated by direct reaction of these two species (39Kono Y. Biochem. Mol. Biol. Int. 1995; 36: 275-283Google Scholar, 41Klebanoff S.J. Free Radical Biol. & Med. 1993; 14: 351-360Google Scholar). Our findings suggest, then, that the reaction product, Cl-NO2, is a strongly oxidizing species that may serve as an antimicrobial agent in its own right.An analogous reaction between hypobromous acid, a product formed by oxidation of Br− catalyzed by eosinophil peroxidase (64Weiss S.J. Test S.T. Eckmann C.M. Roos D. Regiani S. Science. 1986; 234: 200-203Google Scholar), and NO−2 forming Br-ONO and/or Br-NO2 can be envisioned. In fact, Reaction 6 may represent a general mechanism by which hypohalous acids (HOX) react with NO−2 to produce species capable of oxidizing biological molecules. HOX+NO2−→X−ONO+X−NO2+OH− REACTION 6Collectively, these reaction pathways could therefore represent an important host defense mechanism and a novel pathway for inflammation-mediated tissue injury.ConclusionsWe report here that NO−2 and HOCl react to form the reactive intermediates Cl-NO2 and/or Cl-ONO, species that are capable of nitrating, chlorinating, and dimerizing phenolic compounds such as tyrosine. Our data suggest that NO2-Tyr is not necessarily a specific marker of ONOO− formation in vivo and that Cl-NO2 and Cl-ONO may be important and previously unconsidered oxidants produced at sites of inflammation.Note added in proofUpon further investigation of NO+2-mediated nitration of MPA using HPLC, we were unable to detect nitration by the nitryl salt NO2BF4 under neutral aqueous conditions. Hence, we cannot rule out the contribution of an NO+2 species to nitration events observed with NO−2/HOC1 or C1-NO2 in our experiments reported herein. INTRODUCTIONNitrogen monoxide (nitric oxide, ·NO) 1The abbreviations used are: ·NOnitric oxideO2superoxideHOClhypochlorous acidNO−2nitriteONOO−peroxynitriteONOOHperoxynitrous acidNO−3nitrateNO2-Tyr3-nitrotyrosineCl-Tyr3-chlorotyrosineHPA4-hydroxyphenylacetic acidNO2-HPA3-nitro-4-hydroxyphenylacetic acidCl-HPA3-chloro-4-hydroxyphenylacetic acidCl-PhechlorophenylalanineNATN-acetyl-L-tyrosineNAPN-acetyl-L-phenylalanineMPA4-methoxyphenylacetic acidCl-NO2nitryl chlorideCl-ONOchlorine nitriteROSreactive oxygen speciesRNSreactive nitrogen speciesHPLChigh pressure liquid chromatographyPDAphotodiode array. is produced by a variety of cells through the activity of constitutive and inducible forms of nitric oxide synthase (1Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Google Scholar). ·NO is an important endogenous mediator in such diverse biochemical and physiological processes as neurotransmission, smooth muscle relaxation, platelet aggregation and adhesion, macrophage-mediated cytotoxicity, and learning and memory (2Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142Google Scholar, 3Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Google Scholar). Although basal levels of free ·NO are normally quite low (nanomolar), local ·NO concentrations have been shown to increase to levels ranging from 4 to 30 µM under pathologic conditions (4Hooper D.C. Ohnishi S.T. Kean R. Numagami Y. Dietzschold B. Koprowski H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5312-5316Google Scholar, 5Malinski T. Zhang Z.G. Chopp M. J. Cerebral Blood Flow Metab. 1993; 13: 355-358Google Scholar).·NO reacts at a near diffusion-controlled rate with superoxide (O2) (k = 6.7 × 109M−1 s−1) (6Huie R.E. Padmaja S. Free Rad. Res. Commun. 1993; 18: 195-199Google Scholar) to form the cytotoxic species peroxynitrite (ONOO−). The formation of ONOO− is thought to be responsible, at least in part, for the observed toxicity associated with ·NO (7Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Google Scholar, 8Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Google Scholar). At physiological pH the protonated form of ONOO−, peroxynitrous acid (ONOOH) (pKa = 6.8), is highly unstable and rapidly decomposes to nitrate (NO−3). ONOOH is thought to 1) react directly with biological molecules via a vibrationally excited intermediate (ONOOH*), 2) decompose by homolytic dissociation to form nitrogen dioxide (·NO2) and the hydroxyl radical (·OH), or 3) by heterolytic dissociation to form the nitryl cation (nitronium ion, NO+2) (reviewed in Ref. 9Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722Google Scholar). ONOO−/ONOOH reacts with proteins, leading to the oxidation of cysteine, methionine, and tryptophan residues, and can induce protein carbonyl formation and nonspecific fragmentation (10Ischiropoulos H. Al-Mehdi A.B. FEBS Lett. 1995; 364: 279-282Google Scholar, 11Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Google Scholar, 12Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar). In addition, ONOO−/ONOOH can react readily with phenolic compounds to form nitrated, hydroxylated, and dimerized products (13Halfpenny E. Robinson P.L. J. Chem. Soc. (Lond.). 1952; : 939-946Google Scholar, 14Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Google Scholar, 15Van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Google Scholar, 16Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Google Scholar, 17Van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Google Scholar), and nitration of free tyrosine, or tyrosine in proteins, has served as a “marker” and “index” of ONOO− formation in vivo.Based upon tyrosine nitration assays and the formation of “peroxynitrite-specific” luminescence, stimulated macrophages (18Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Google Scholar), neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar), and endothelial cells (20Kooy N.W. Royall J.A. Arch. Biochem. Biophys. 1994; 310: 352-359Google Scholar) have been proposed to form significant quantities of ONOO− in vitro. In fact, the detection of 3-nitrotyrosine (NO2-Tyr) in a variety of pathologic conditions in vivo, such as inflammatory lung disease (21Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Google Scholar), atherosclerosis (22Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biochem. Hoppe-Seyler. 1994; 375: 81-88Google Scholar), and rheumatoid arthritis (23Kaur H. Halliwell B. FEBS Lett. 1994; 350: 9-12Google Scholar), has been attributed to ONOO− formation. However, in all of these cases direct proof for the production of ONOO− in biological systems is lacking, even though its formation in vivo is favorably predicted (24Squadrito G.L. Pryor W.A. Chem. Biol. Interact. 1995; 96: 203-206Google Scholar).Under inflammatory conditions, multiple well characterized reactive oxygen species (ROS) are produced from phagocytic cells (25Miller R.A. Britigan B.E. J. Invest. Med. 1995; 43: 39-49Google Scholar). For instance, stimulated neutrophils and macrophages produce significant levels of superoxide (O2) and hydrogen peroxide (H2O2) as a result of the activation of the respiratory burst oxidase (26Winterbourn C.C. Das D.K. Essman W.B. Oxygen Radicals: Systemic Events and Disease Processes. Karger, Basel, Switzerland1990: 31Google Scholar). In the case of neutrophils, some of the H2O2 that is produced under these conditions is converted to the strong oxidant hypochlorous acid (HOCl) by the action of myeloperoxidase as shown in Reaction 1. H2O2+H++C1−→HOC1+H2O REACTION 1HOCl produced from activated human neutrophils has been shown to react with amines (taurine, lysine, and arginine) and tyrosine to form N-chloramines and 3-chlorotyrosine (Cl-Tyr), respectively (27Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Google Scholar, 28Weiss S.J. Lampert M.B. Test S.T. Science. 1983; 222: 625-628Google Scholar), where the latter has been proposed to serve as a selective marker of HOCl production in vivo (29Kettle A.J. FEBS Lett. 1996; 379: 103-106Google Scholar).In addition to ROS, macrophages (30Marletta M.A. Yoon P.S. Iyengar R. Leaf C.D. Wishnok J.S. Biochemistry. 1988; 27: 8706-8711Google Scholar) and neutrophils (19Carreras M.C. Pargament G.A. Catz S.D. Poderoso J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Google Scholar) can also simultaneously produce large fluxes of ·NO through the activation of inducible nitric oxide synthase; however, the ability of human neutrophils to produce ·NO is debated (31Miles A.M. Owens M.W. Milligan S. Johnson G.G. Fields J.Z. Ing T.S. Kottapalli V. Keshavarzian A. Grisham M.B. J. Leukocyte Biol. 1995; 58: 616-622Google Scholar). Once formed, ·NO can react with several biological targets, primarily thought to involve heme-iron, hyperreactive sulfhydryls and protein radicals (32Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar, 33Lepoivre M. Flaman J.-M. Bobé P. Lemaire G. Henry Y. J. Biol. Chem. 1994; 269: 21891-21897Google Scholar, 34Eiserich J.P. Butler J. van der Vliet A. Cross C.E. Halliwell B. Biochem. J. 1995; 310: 745-749Google Scholar). ·NO can also react with O2 in aqueous solution to produce nitrite (NO−2) via a complex mechanism thought to involve a variety of reactive nitrogen species (RNS) including ·NO2 and dinitrogen trioxide (N2O3) (35Ignarro L.J. Fukuto J.M. Griscavage J.M. Rogers N.E. Byrns R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8103-8107Google Scholar). In fact, NO−2 has been used as a marker of ·NO production in vitro and in vivo and has been shown to reach concentrations of up to 4 µM in synovial fluid from patients with rheumatoid arthritis (36Farrell A.J. Blake D.R. Palmer R.M.J. Moncada S. Ann. Rheum. Dis. 1992; 51: 1219-1222Google Scholar) and as high as 20 µM in human airway fluids (37Gaston B. Reilly J. Drazen J.M. Fackler J. Ramdev P. Arnelle D. Mullins M.E. Sugarbaker D.J. Chee C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10957-10961Google Scholar). These RNS produced during an inflammatory response could theoretically react with a number of ROS to form various novel species. Indeed, the interaction of HOCl with ·NO or NO−2 has been proposed to form species capable of nitrosylating and nitrating organic substrates (38Koppenol W.H. FEBS Lett. 1994; 347: 5-8Google Scholar, 39Kono Y. Biochem. Mol. Biol. Int. 1995; 36: 275-283Google Scholar).The present study was undertaken to examine the potential interactions of ·NO-derived RNS with the inflammatory oxidant HOCl in an attempt to characterize more fully the various species that may be formed under complex physiological inflammatory conditions. Our results indicate that NO−2 reacts with HOCl to form an intermediate species, postulated to be nitryl chloride (Cl-NO2) and/or chlorine nitrite (Cl-ONO), that is capable of nitrating, chlorinating, and dimerizing phenolic compounds including tyrosine. We propose that the formation of Cl-NO2 and/or Cl-ONO by this reaction represents a novel mechanism of inflammation-mediated biological damage, and offers an additional or alternative mechanism of tyrosine nitration independent of ONOO− formation.