Nitric Oxide, Oxidants, and Protein Tyrosine Nitration

PERSPECTIVE

Nitric oxide, oxidants, and protein tyrosine nitration

Rafael Radi* Departamento de Bioquı´micaand Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Repu´blica, Avda. General Flores 2125, 11800 Montevideo, Uruguay

Communicated by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, January 12, 2004 (received for review November 12, 2003)

The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the signal transducing physiological actions of •NO to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive ni- • • ؊ trogen species such as peroxynitrite anion (ONOO ) and nitrogen dioxide ( NO2), formed as secondary products of NO metabolism in the ؊• presence of oxidants including superoxide radicals (O2 ), hydrogen peroxide (H2O2), and transition metal centers. The precise interplay between •NO and oxidants and the identification of the proximal intermediate(s) responsible for nitration in vivo have been under contro- versy. Despite the capacity of peroxynitrite to mediate tyrosine nitration in vitro, its role on nitration in vivo has been questioned, and alternative pathways, including the nitrite͞H2O2͞hemeperoxidase and transition metal-dependent mechanisms, have been proposed. A balanced analysis of existing evidence indicates that (i) different nitration pathways can contribute to tyrosine nitration in vivo, and (ii) ؊• most, if not all, nitration pathways involve free radical biochemistry with carbonate radicals (CO3 ) and͞or oxo–metal complexes oxidizing • tyrosine to tyrosyl radical followed by the diffusion-controlled reaction with NO2 to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine has been revealed as a relevant biomarker of •NO-dependent oxidative stress; additionally, site-specific nitration focused on particular protein tyrosines may result in modification of function and promote a biological effect. Tissue distribution and quantitation of protein 3-nitrotyrosine, recognition of the predominant nitration pathways and individual identification of nitrated proteins in disease states open new avenues for the understanding and treatment of human pathologies.

arly after the discovery of the sig- and chronic disease states, and can be a ute to protein tyrosine nitration. Interest- nal transducing physiological func- predictor of disease risk. ingly, the alternative nitration pathways tions of the free radical nitric ox- Seminal work by Beckman et al. (6) and share common characteristics, because Eide (•NO) in the vasculature and Ischiropoulos et al. (7) demonstrated the they involve free radical biochemistry with nervous system (e.g., vasodilation and capacity of peroxynitrite to cause protein the participation of transient tyrosyl, • •Ϫ neurotransmission), it became evident that tyrosine nitration in vitro and established NO2, and carbonate (CO3 ) radicals •NO could also participate as a cytotoxic the concept that biologically produced and͞or oxo–metal complexes. effector molecule and͞or a pathogenic intermediates could promote nitration in mediator when produced at high rates by vivo (8, 9), as was also suggested in an NO Reaction with Superoxide and the either inflammatory stimuli-induced nitric earlier work by Ohshima et al. (10). How- Formation of Peroxynitrite oxide synthase (iNOS) or overstimulation ever, the role of peroxynitrite as a central A relevant debate in the field has been of the constitutive forms (eNOS and species in biological nitration has been whether peroxynitrite can be produced nNOS) (1). Much of •NO-mediated more recently questioned (11, 12), and biologically at levels high enough to play a pathogenicity depends on the formation alternative pathways, most notably mecha- significant role in •NO-dependent pathol- of secondary intermediates such as per- nisms that depend on the formation of ogy (16, 17). Because peroxynitrite is a Ϫ • oxynitrite anion (ONOO ) and nitrogen NO2 by the action of hemeperoxidases transient species with a biological half-life • ͞ dioxide ( NO2) that are typically more and or transition metal complexes on a [10–20 ms (18)] even shorter than that of reactive and toxic than •NO per se (2). main product of •NO metabolism, nitrite •NO [1–30 s (1)], it cannot be directly Ϫ The formation of reactive nitrogen species (NO2 ), have been presented as contribu- measured, and its presence must be in- from •NO requires the presence of oxi- tors (12–15). ferred from a combination of analytical, •Ϫ ͞ dants such as superoxide radicals (O2 ), In addition to nitration, inflammatory pharmacological, and or genetic ap- † hydrogen peroxide (H2O2), and transition conditions promote other oxidative modi- proaches (19). The biological reactions of •Ϫ metal centers, the concentration of which fications in tyrosine such as chlorination, NO with O2 were initially proposed dur- can be increased either by •NO itself or bromination, and hydroxylation to ing studies that characterized the chemical by the same mediators that up-regulate 3-chloro-, 3-bromo-, or 3-hydroxytyrosine, nature of the endothelial-derived relaxing •NO production. Nitrogen dioxide can the detection of which may assist in iden- factor as •NO (20–22). Indeed, the bio- also be formed in hydrophobic environ- tifying preferential nitration pathways. logical half-life and actions of •NO in the ments from the reactions of •NO with The extent of these oxidative modifica- vasculature were prolonged by superoxide molecular oxygen, where these species tions in vivo can reach similar values per dismutase (SOD), the enzyme that elimi- •Ϫ concentrate (3, 4). One of the molecular milligram of tissue protein (e.g., 10–100 nates O2 at diffusion-controlled rates, footprints left by the reactions of reactive pmol͞mg) and are comparable to the lev- nitrogen species with biomolecules is the els of protein S-nitrosation. It is also im- nitration (i.e., addition of nitro group, portant to appreciate that other oxidative Abbreviations: NOS, NO synthase; MPO, myeloperoxidase; Ϫ EPO, eosinophil peroxidase; SOD, superoxide dismutase; NO2) of protein tyrosine residues to processes triggered by reactive nitrogen LMW, low molecular weight. 3-nitrotyrosine. The formation of protein species such as thiol and methionine oxi- *E-mail: [email protected]. 3-nitrotyrosine was originally addressed in dation, disruption of iron–sulfur clusters, †The debate on the biological production and measure- early protein chemistry studies with tetra- and oxidation of transition metal centers ment of peroxynitrite is analogous to a previous one on •Ϫ nitromethane aimed at establishing the (2) can, in many cases, be more relevant O2 that arose after the discovery of SOD by J. M. McCord function of tyrosines in proteins (5). This than nitration in the promotion cell dys- and I. Fridovich. The recurring discussions during the ﬁrst ͞ decade led I. Fridovich and associates to invest large re- now-established posttranslational modifi- function death. search efforts into fully (and redundantly) establishing the •Ϫ cation attracts considerable interest to A balanced analysis of all of the exist- concept that O2 was a biologically relevant reactive in- biomedical research, because it can alter ing in vitro and in vivo evidence indicates termediate. protein function, is associated to acute that more than one pathway can contrib- © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307446101 PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 ͉ 4003–4008 Downloaded by guest on September 28, 2021 •Ϫ Ϫ1 Ϫ1 and were decreased by enhanced O2 for- The Transition Metal-Dependent M s ) oxo–iron intermediates (14, 15) mation by redox-cycling molecules or high Formation of Strong Oxidants and (Eqs. 3–5): oxygen tensions. Thus, early data firmly Nitrogen Dioxide Ϫ ͑ 3ϩ͒ ϩ 3 • • MPO Fe H2O2 established the existence of NO and O2 Elevations of redox active metals systems •␲ϩ ϩ interactions, although at the time the re- such as iron (Fe), copper (Cu), and man- MPO ͑Fe4 ͒͑compound I͒ [3] action was perceived as one to just limit ganese (Mn) contribute to oxidative dam- the biological half-life of •NO to yield •␲ϩ ͑ 4ϩ͒ ϩ Ϫ 3 Ϫ age in disease states (12, 14). Also, in- MPO Fe NO2 relatively inert nitrate (NO3 ). flammatory conditions trigger the release ͑ 4ϩ͒ ϩ • ͑ ͒ The rate constant of the reaction of of leukocyte peroxidases, including myelo- MPO Fe NO2 compound II •Ϫ • ϳ 10 Ϫ1 Ϫ1 O2 with NO is larger ( 10 M s ) peroxidase (MPO) and eosinophil peroxi- Ϫ Ϫ [4] than with SOD (1–2 ϫ 109 M 1s 1), and dase (EPO), which play central roles in • tissue oxidant formation (13, 15, 41). Oxi- ͑ 4ϩ͒ ϩ Ϫ 3 therefore NO sometimes outcompetes MPO Fe NO2 •Ϫ • SOD for O2 . The reaction of NO with dative stress promotes metal mobilization •Ϫ ͑ 3ϩ͒ ϩ • O2 leads to the diffusion-controlled for- from proteins by mechanisms that include MPO Fe NO2 [5] Ϫ •Ϫ mation of peroxynitrite anion (ONOO ) O2 -mediated oxidation of labile iron– (Eq. 1; for a review, see ref. 2): sulfur clusters [e.g., aconitase and other Other hemeproteins, free hemin, and dehydratases (42)], redox-dependent LMW complexes can undergo similar re- Ϫ Ϫ • • ϩ • 3 actions to produce NO2, although pre- NO O2 ONOO [1] metal release from storage or transport proteins (transferrin, ferritin, and cerulo- sumably at lower rates (12). The influence of SOD on the half-life of plasmin), oxidative modifications of heme The reactions of peroxynitrite with oxi- •NO and the formation of peroxynitrite dized transition metal centers including Ϫ proteins that result in heme release under various fluxes of •NO and O• (23) ͞ MPO (45) can sometimes concomitantly 2 and or degradation (myoglobin and cyto- • yield oxo–metal complexes and NO2. In- and in different vascular tissue layers (24) chrome c), and histidine oxidation with Ϫ •Ϫ deed, the OOO bond in ONOO is sig- have been recently analyzed. Being O2 , disruption of metal coordination sites (i.e., nificantly weakened by association with one of the factors controlling the half-life, Cu-Zn SOD), among others. Metals re- ϩ for instance, of vascular- (20–22) and in- leased from proteins are trapped by low Lewis acids such as H ,CO2, or transition metal centers (MenX), which favor its ho- flammatory cell- (25) derived •NO, the molecular-weight (LMW) chelators such molysis (ref. 46; Eq. 6): subsequent formation of peroxynitrite is as adenine nucleotides (ATP3-and ADP2-), Ϫ ‡ • • Ϫ obligatory. However, NO and O2 reac- tri- and dicarboxylic acids (citrate, isocit- ONOO ϩ MenX 3 tions do not necessarily result in tissue rate, and piruvate), or phosphate or bind n 3 • ϩ • n 3 oxidative injury and in some cases can to macromolecules and in conjunction ONOO–Me X NO2 O–Me X even be cytoprotective (30, 31). Indeed, with free hemin (12, 14) become more • ϩ ϭ nϩ1 low levels of peroxynitrite could be detox- readily accessible to undesired redox bio- NO2 O Me X [6] ified by enzymatic and nonenzymatic sys- chemistry (43). Typically, in the reductive ϩ Although the H -orCO-catalyzed ho- tems (32–35) and then, direct toxic effects environment of tissues, these metal com- 2 Ϫ molysis results in radical yields of Ϸ30% • • plexes become reduced, and in particular of either NO or O2 neutralized. Forma- and 35%, respectively, oxidant yield from ferrous iron can react with hydrogen per- tion of peroxynitrite might also play subtle metals (Eq. 6) can, in some cases, reach oxide to form variable amounts of free roles in signal transduction processes (36– up to 100% (6, 46). Thus, there is positive •OH and oxoferryl species [Fenton reac- 39). However, identification of oxidation feedback among redox active metals and tion (43); Eq. 2]: and nitration products in conjunction with oxidants and •NO to facilitate the produc- ͞ pharmacological and or genetic ap- ϩ tion of the mediators of nitration. X-Fe3 ϩ •OH [2a] proaches has substantiated direct toxicity m 2ϩ ϩ Iron Regulatory Proteins, •NO, and of peroxynitrite in pathophysiologically X-Fe H2O2 relevant conditions (see ref. 19 and refer- Oxidant Stress n ences therein and below). Peroxynitrite is NO and oxidants influence the biochemi- 4ϩ a strong one- and two-electron oxidant X-Fe ϭO [2b] cal mechanisms that regulate cell iron ho- • • and can also evolve to OH, NO2, and meostasis and that involve iron regulatory Ϫ • CO• radicals. Peroxynitrite does not re- The large reactivity of free OH with proteins 1 and 2 (IRP-1͞2). IRPs play 3 most biomolecules and its minimal diffu- act directly with tyrosine (2) but oxidizes pivotal roles in assuring the maintenance sion distance (three to four molecular di- and nitrates it through its radical products. of iron in the LMW pool at levels suffi- ameters) makes it unselective for protein Revisions on the biological chemistry of cient for the biosynthesis of heme- and modification unless site-specifically formed peroxynitrite and its derived radicals have nonheme proteins but not exceeding within metal-binding sites; oxo–iron com- been published recently (2, 19, 40). amounts that may impose a risk of metal- plexes live longer and are more selective dependent oxidative damage (47). IRP-1 ͞ in substrate target reactions. Oxidized in its inactive state contains a cubane transition metals also react with H2O2 to 4Fe-4S cluster with a labile iron (Fe␣) ‡Under some circumstances, SOD could augment the bio- form secondary oxidizing species, e.g., and displays aconitase activity. Total dis- • •Ϫ availability of NO by O2 -independent mechanisms such ferric heme reaction with H2O2 yields an assembly of the cluster to the apoprotein as (i) the SOD-mediated oxidation of nitroxyl anion (NOϪ) oxoferryl intermediate plus a porphyrin yields a fully active IRP-1, which in turn • to NO (26);. and (ii) the SOD-mediated reduction of S- radical cation (14). promotes down-regulation of ferritin and nitrosothiols (27). These alternative mechanisms for the actions of SOD could mainly operate intracellularly (28). Oxo–metal complexes, in turn, favor up-regulation of the transferrin receptor • Ϫ The immediate vascular responses to extracellularly added formation of NO2. Indeed, NO2 evolves with an overall increase of LMW iron. • • SOD are due to the prevention of the oxidative inactiva- to NO2 via a one-electron oxidation. For Mild oxidative stress and͞or NO levels • ͞ •Ϫ tion of readily diffusible NO. NO O2 interactions in the instance, MPO (and EPO) can oxidize vasculature are viewed as contributions to the develop- lead to IRP-1 activation, whereas over- Ϫ ϭ ϫ 6 ment of vascular disease states, including atherogenesis, NO2 by compounds I (k 2 10 production of reactive oxygen species or Ϫ Ϫ hypertension, and hyperglycemia (29). M 1s 1) (44) and II (k ϭ 5.5 ϫ 102 peroxynitrite extends their action beyond

4004 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307446101 Radi Downloaded by guest on September 28, 2021 ϩ Then, the polarized carrier or NO2 itself could attack tyrosine as a two-electron acceptor to yield a nitroarenium ion intermediate, which then evolves to 3-nitrotyrosine and a proton (50) (Eqs. 8–10):

n ϩ 3 NO2–O–Me X TyrH ͓ n ͔ 3 TyrH-NO2–O–Me X ϩ ϭ n ϩ ϩ NO2–Tyr O Me X H [8] ϩ ϩ 3 NO2 TyrH ͓ ϩ͔ 3 ϩ ϩ NO2–TyrH NO2–Tyr H [9] ϭ n ϩ ϩ 3 n ϩ O Mn X 2H Me X H2O [10] ϩ The half-life of NO2 is too short in aque- Fig. 1. The free radical pathways of tyrosine nitration. ous systems because of its fast reaction Ϫ with H2O and subsequent decay to NO , ϩ 3 and therefore the metal-bound NO2 com- reversible cluster disassembly and pro- thiols. The dimerization of tyrosyl radicals plex would be the proximal reactant (Eq. Ј duces irreversible inactivation via oxida- to 3,3 -dityrosine competes with the for- 8). This mechanism of nitration§ may op- tion of critical thiols. IRP-2 does not con- mation of 3-nitrotyrosine. However, pro- erate for some metal complexes such as in tain the iron–sulfur cluster, and its activity tein tyrosyl radicals can be stabilized, with the peroxynitrite-dependent nitrations of is controlled by level of expression and intra- and intermolecular dimerization Tyr-108 of bovine CuZn SOD (6) or oxidant-stimulated proteolytic limited due to spatial and diffusional con- Tyr-34 of human MnSOD (51), although degradation. straints, both in aqueous and hydrophobic current data cannot rule out a free radical The oxidant-dependent inactivation of compartments, in which case their reac- mechanism. • IRPs may be viewed as a negative feed- tion with NO2 is favored. Another com- Electrophilic aromatic nitration can also back to inhibit the amplification of oxida- peting pathway is the formation of 3-hy- occur in vitro by the action of nitryl chlo- tive damage but, in fact, it disrupts the droxytyrosine, which can be mediated by ride (NO2Cl), formed from the relatively reversible mechanisms for controlling iron •OH and oxo–metal complexes (15). An slow reaction of MPO-derived hypochlor- homeostasis and may render the cell more Ϫ alternative radical mechanism for tyrosine ous acid with NO2 . However, the role of vulnerable. Also, an increase of the labile nitration involves the reaction of a tyrosyl • NO2Cl as a nitrating molecule in vivo is iron pool by NO may favor, on initiation radical with •NO to form 3-nitrosotyro- highly unlikely (52). In summary, present of oxidant stress, nitration reactions in sine followed by a sequential two-electron evidence cannot yet confirm that electro- target cells before the homeostatic re- oxidation to 3-nitrotyrosine via tyrosine philic aromatic nitration is a biologically sponses for iron sequestration at the pro- iminoxyl radical; this mechanism may relevant mechanism. tein expression level are achieved. Inter- operate in transition metal-containing estingly, during inflammatory conditions, proteins that can readily oxidize The Limited Efficiency of the Nitration IRP-1 is found to be not only inactivated 3-nitrosotyrosine before the latter can Reactions in Biology but also nitrated (48). The role of IRPs in reversibly generate tyrosyl radicals and Biological nitration yields are low, and, the regulation of iron-dependent nitration •NO, such as in the case of prostaglandin under inflammatory conditions, one to reactions remains to be elucidated. H synthase-2 (49). five 3-nitrotyrosine residues per 10,000 tyrosine residues (100–500 ␮mol͞mol) are The Free Radical Pathways to Is Electrophilic Aromatic Nitration a Tyrosine Nitration detected (15, 53). This is due to a multi- Biological Mechanism? plicity of processes (reactions, diffusion) The very existence of 3-nitrotyrosine in The reactions of peroxynitrite with transi- that the precursors of nitrating species vivo supports free radical biochemistry •Ϫ • tion metal centers may promote tyrosine (O2 ,H2O2, NO) and the proximal in- (Fig. 1). In fact, the two most widely in- • •Ϫ nitration by a free radical-independent termediates of nitration ( NO2,CO , voked mechanisms of biological nitration, 3 chemistry, namely electrophilic aromatic oxo–metal complexes) can undergo, namely the peroxynitrite and the heme- nitration (6, 7). In this mechanism, per- to repair mechanisms of the tyrosyl peroxidase pathways, lead to the concomi- oxynitrite would first form a complex with radical such as reduction by glutathione tant formation of tyrosyl radicals and the transition metal to yield a polarized and, presumably, by metabolism of •NO , which combine at diffusion- ϩ 2 carrier of nitronium cation (NO ), which 3-nitrotyrosine.¶ controlled rates to form 3-nitrotyrosine 2ϩ may then decompose to free NO by het- (Fig. 1). 2 erolysis (Eq. 7): The oxidants leading to tyrosyl radical §In the electrophilic aromatic nitration mechanism, there is Ϫ • Ϫ are either CO3 or oxo–metal complexes ONOO ϩ MenX 3 no net redox change in the metal center (Eqs. 7–10). How- and, to a lesser extent, •OH. Importantly, ever, direct and unambiguous spectroscopic observation is sometimes difﬁcult due to low steady-state concentrations • n 3 NO2 alone is inefficient in promoting ONOO–Me X and͞or the absorption of peroxynitrite–metal complexes; • nitration, because it must react first with additionally, there is no formation of NO2. ␦ϩ ␦Ϫ NO –O –MenX 3 tyrosine to yield tyrosyl radical, a reaction 2 ¶Nitrated proteins can also undergo enhanced proteolytic that is slow compared to other processes ϩ degradation (54), and reports have suggested that a deni- • ϩ ϭ n that NO2 undergoes, for example, with NO2 O Me X [7] trase activity may be present in vivo (55).

Radi PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 ͉ 4005 Downloaded by guest on September 28, 2021 The idea of peroxynitrite as a mediator dases may serve as catalytic sinks of •NO that are more superficial than the metal- of biological nitration was challenged by and influence nitration yields (64). adjacent Tyr-34, and that the association observations demonstrating that the oxi- of nitration plus inactivation of MnSOD dative biochemistry of peroxynitrite could The Biological Significance of Protein may serve to unravel the chemical nature be strongly modulated by the relative Tyrosine Nitration of the nitrating species. A recent quantita- • •Ϫ fluxes of NO and O2 (56–58). Indeed, The small fraction of nitrated protein in tion of the extent of MnSOD nitration in in homogenous systems, reasonable nitra- the context of total tissue protein ques- rat kidneys during angiotensin II infusion tion yields are obtained only at an optimal tions its possible biological relevance. It is provided support for the hypothesis of • ͞ •Ϫ ϳ NO O2 flux ratio one (11, 58), be- noteworthy to indicate, however, that (i)a nitration as a direct cause of enzyme inac- • •Ϫ cause excess of either NO or O2 reacts relatively limited number of proteins are tivation (71). Similarly, the vascular en- • with tyrosyl radicals and NO2 and deacti- preferential targets of nitration, and (ii) zyme prostacyclin synthase (PGI2) syn- vates the process. Also, under low radical within these proteins, only one or a few thase becomes site-specifically nitrated by fluxes, the direct reaction of tyrosine with specific tyrosines can be nitrated (65); • peroxynitrite at heme-adjacent Tyr-430 in NO2 competes with the recombination because the species participating in nitra- • a process catalyzed by the active site reaction of NO2 with the tyrosyl radical. tion have short diffusion distances and heme-thiolate involving transient ferryl- The observations demonstrated that free site-specific nitration occurs at metal- or species (72, 73). Note that the subcellular radical processes triggered by pure per- hemeperoxidase-binding sites, nitration distribution of PGI synthase in vascular oxynitrite may result in outcomes different 2 reactions can be concentrated on proteins, endothelium, colocalized with eNOS at than those initiated by the simultaneous Ϫ cell, or tissue compartments as revealed production of •NO and O• (11, 58) do the caveola, may further contribute for it 2 by immunochemical (with antibodies to be a sensitive and critical target of per- not reconcile well with solid biological against protein 3-nitrotyrosine) and pro- ʈ evidence (see below), supporting peroxynitrite. Recent experiments indicate teomic-based methods. Thus, although that inflammation of artery walls results oxynitrite-mediated nitration in vivo. This there is a dilutional effect in quantitating apparent conflict can be solved by appre- in rapid PGI2 synthase nitration by a per- total nitrated protein by analytical tech- oxynitrite-dependent mechanism (73). ciating the role of two key factors present niques in tissues, nitration can be focused Examples of MnSOD and PGI2 synthase in cells and tissues: first, SOD, which sig- on specific tyrosines and potentially result nificantly lessens increases in the steady- affirm the concept that transition metal •Ϫ in modification, loss, or gain of function. catalysis provides selectivity and efficiency state levels of O2 , despite large varia- Ϫ •Ϫ Addition of a NO2 group to tyrosine tions of O2 production rates; and Ϫ to peroxynitrite as a biological nitrating lowers the pKa of its phenolic OH by agent. Finally, it is relevant to note that second, the rapid transmembrane diffu- 2–3 units and adds a bulky substituent; if sion and͞or cell consumption of •NO that actin, which can constitute 5% or more of placed in relevant tyrosines, nitration can operates as a drain of excess •NO; thus, cell protein, is heavily nitrated in sickle Ϫ alter protein function and conformation, augmentation of either •NO or O• cell disease and that the extents of nitra- 2 impose steric restrictions, and also inhibit fluxes in vivo will serve to trap more of tion observed in tissues are sufficient to tyrosine phosphorylation. However, to the partner radical, which otherwise is induce defective cytoskeletal polymeriza- have biological significance, a loss-of-func- being consumed by other processes in- tion (53). tion modification requires a large fraction stead of accumulating and interfering with The clinical relevance of protein ty- of protein to become nitrated at specific the nitration process. Experimental con- rosine nitration has been recently under- critical tyrosines and result in 3-nitroty- founding factors have also obscured this scored by the observation of a strong as- discussion, including, in some works, the rosine to tyrosine ratios in the range of 0.1–1.0; documentation of these large ra- sociation between protein 3-nitrotyrosine presence of uric acid (see below) and the levels and coronary artery disease risk lack of consideration of the diffusional tios for a given protein in vivo is scanty, and it is doubtful that many proteins will (75). Circulating levels of protein 3-nitro- properties of extracellular peroxynitrite tyrosine may serve as a biomarker to as- •Ϫ undergo such an extent of nitration. An and CO3 . Thus, a modest nitration but sess atherosclerosis risk as well as to mon- one that is responsive to the increase on alternative scenario is a gain-of-function Ϫ itor the vasculoprotective action of drugs. • ͞ • modification, in which case a small frac- the absolute NO and or O2 production Interestingly, circulating MPO levels may rates can be generated by peroxynitrite in tion of nitrated protein can elicit a sub- stantive biological signal. This attractive also serve as predictor of cardiovascular vivo. As examples at the cell level, the role • concept has been shown in a few proteins risk (76). Thus, enhanced NO consump- of endogenous peroxynitrite in protein tion by either vascular wall͞inflammatory nitration has been substantiated by thor- such as cytochrome c, which acquires a •Ϫ cell-derived O and͞or MPO provides a ough studies on motoneurons (59) and strong peroxidase activity after nitration 2 common link between decreases in •NO smooth muscle cells (60) undergoing bio- (66), in nitrated fibrinogen, which acceler- bioavailability and increases in protein logically relevant challenges. ates clot formation (67) and in protein ␧ In inflammatory cells, the hemeperoxi- kinase C , which becomes activated and tyrosine nitration. translocates on nitration (68). dase nitration pathway (MPO, EPO) ap- Mitochondrial Nitration and Its A notable example of loss of enzyme pears to more important extra- than intra- Relevance to Cell Death cellularly, because intracellular activity linked to nitration in vivo is the peroxidases may have limited supply of mitochondrial enzyme MnSOD. This pro- Mitochondria have been recognized as Ϫ critical sources and targets of nitrating NO2 (61, 62). However, after polymor- tein is nitrated by peroxynitrite in Tyr-34 phonuclear cell degranulation, MPO can by a Mn-catalyzed process, which leads to species (reviewed in ref. 77). Although the • be taken up by nonphagocytic cells, and a enzyme inactivation. Nitrated and inacti- formation of NO mainly occurs at extra- strong codistribution of MPO and 3-nitro- vated MnSOD is found in acute and mitochondrial sites, the facile diffusion of tyrosine is observed in different inflam- chronic inflammatory processes both in •NO to mitochondria and its combination matory processes (63). Excess •NO can animal models and human diseases (69).

also modulate the hemeperoxidase-medi- Interestingly, MnSOD nitration via the ʈ • ͞• Some metabolic conditions favor the role of NOS as a ated nitration; indeed, NO readily reacts hemeperoxidase NO2 pathway does not signiﬁcant source of peroxynitrite (74). NOS, in turn, can with resting state MPO as well as with lead to significant inactivation (70), sug- be nitrated and potentially inactivated both in vitro and compounds I and II, and therefore peroxi- gesting that nitration occurs at tyrosines in vivo.

4006 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307446101 Radi Downloaded by guest on September 28, 2021 •Ϫ with mitochondrial-derived O2 results in respiratory chain uncouplers, among jury and nitration in neurotoxicity and the formation of peroxynitrite, which ac- others. If the mechanism is peroxynitrite- vascular injury models (92, 93).** On the counts for much of the disruption of mito- dependent, it should be inhibited SOD contrary, heterozygous MnSOD knock- chondrial metabolism initially attributed (native, modified, or liposome-entrapped) outs resulted in larger extents of brain to direct actions of •NO and to the nitra- or SOD-mimics, as long as the enzyme is nitration in a model of neurotoxicity, con- tion of mitochondrial proteins. A cyto- located in the same compartment where Ϫ sistent with enhanced mitochondrial for- chrome c-dependent peroxidase-like O2 is formed, and is unaffected by MPO mation and reactions of peroxynitrite (94). mechanism of nitration may also operate inhibitors and catalase (60, 85). In cases Ϫ MPO and EPO knockouts have evi- (70). Mitochondrial proteins are nitrated where SOD is not close enough to O2 denced the peroxidase-dependent nitra- in vitro and in vivo, including MnSOD, sources, it can enhance peroxynitrite- tion pathway in models of inflammation aconitase, cytochrome c, voltage-depen- dependent nitration (7, 8). On the other (15), but the relative contribution was dent anion channel, ATPase, and succi- hand, hemeperoxidase-dependent path- highly model-dependent. The inability of nyl-CoA oxoacid-CoA transferase (77, 78). ways can be stimulated by SOD, inhibited ͞ MPO from human neutrophils to cause The nitration inactivation of MnSOD op- by catalase and MPO inhibitors, as ob- nitration in some models may result from erates as a positive loop for enhanced in- •Ϫ served in H2O2-producing inflammatory lack of H O substrate, because all O tramitochondrial peroxynitrite formation, Ϫ 2 2 2 cells in the presence of NO2 (13). The can react with •NO to form peroxynitrite which in turn triggers apoptotic signaling participation of LMW iron in the nitration (25) and͞or because the nitration capabili- of cell death, in part by the thiol oxida- reactions can be assessed by metal chela- ties of hemeperoxidases may vary accord- tion-dependent assembly of the perme- tors, which block Fenton chemistry, al- Ϫ ability transition pore (77). Detection of ingtotheiraccesstoNO2 and peroxyni- though experiments in this line are scarce. trite (61, 62, 95). In this context, release nitrated mitochondrial proteins and even Importantly, desferrioxamine can also di- Ϫ and sequestration of neutrophil-derived nitrated cytochrome c in the cytosol may • • rectly trap NO2 and CO3 and prevent MPO in specific tissue compartments dur- serve to reflect extents of mitochondrial tyrosine nitration (86). • ing inflammation are important aspects of NO-dependent oxidative stress (66, 77, Uric acid inhibits nitration reactions 78). Importantly, the enhanced peroxi- MPO distribution and nitration capacity in and has been used as a scavenger of per- vivo (63). However, results involving MPO datic activity of nitrated cytochrome c (66, oxynitrite in vitro and in vivo (87). Al- in mice should be cautiously extrapolated 77) may further contribute to oxidative though uric acid does not react at appre- to human inflammatory disease; indeed, damage. ciable rates with peroxynitrite (2), it surprisingly, whereas MPO levels posi- readily reacts with peroxynitrite-derived Red Blood Cell Hemoglobin as a Sink of tively correlate with cardiovascular risk in radicals and oxo–metal complexes. Unfor- Nitrating Species humans (76), MPO knockout mice de- tunately, the presence of uric acid has Not all hemeproteins promote peroxyni- velop atherosclerosis more rapidly than confounded the analysis of nitration yields trite-dependent nitration under biologi- WT (96). MPO or EPO overexpresses are by peroxynitrite in various reports when cally relevant conditions, because some not available yet, but in the future they Ϫ xanthine͞xanthine oxidase has been used reduce it by two electrons to NO or Ϫ Ϫ 2 • may also provide clues to the role of as a source of O2 and can be easily over- isomerize it to NO3 (79–82). In this con- MPO and EPO in pathology. Enhanced text, oxyhemoglobin (oxyHb), present at 5 come with the use of alternative sub- strates (88). LMW porphyrin complexes removal of H2O2 by stable transfection of mM in red blood cells, serves as a sink to cells with catalase can also be useful to capture intravascularly formed nitrating with Mn and Fe have SOD-mimic and peroxynitrite-reductase activities and react assess the contribution of the hemeperoxi- species. Indeed, a fraction of intravascu- •Ϫ dase pathway (60). The hemeperoxidase- larly formed peroxynitrite could diffuse quickly with CO3 (89). They have been successfully used to inhibit •NO-depen- independent nitration observed in MPO into red blood cells before enacting target and EPO knockouts (15) may be due, in molecule reactions with plasma compo- dent injury and nitration in cell and animal models of inflammation (90, 91). The addition to peroxynitrite, to the LMW nents (83) and could undergo a fast reac- metal-dependent pathway (12). The recent tion with oxyHb, which results in its redox and kinetic properties of these Ϫ development of IRPs knockout mice (97) isomerization to NO , without significant SOD-mimics and peroxynitrite decompo- 3 offers opportunities to study inflammatory formation of nitrating species (82). Hemo- sition catalysts can be modulated by models and nitration in the context of globin nitration is observed only under changes in the porphyrin substituents, ͞ excess peroxynitrite over hemoglobin. Be- which can also affect its cellular tissue altered iron homeostasis. • Ϫ distribution, aspects that require further Circulating immune system cells (i.e., cause NO and NO2 are also oxidized by Ϫ research. monocytes and neutrophils) from patients oxyHb to rather unreactive NO3 , oxyHb may ultimately serve to inhibit the intra- suffering from genetic deficiencies of Lessons from Genetics: Knockouts, vascular formation of •NO . A similar NADPH oxidase (chronic granulomatous 2 Transgenics, and Human Deficiency inhibitory role on the formation of disease) or MPO (MPO deficiency) have Syndromes • nitrating species may be played by deoxy- altered interactions of NO with oxidants Ϫ hemoglobin, which reduces NO2 to Knockout and transgenic animals and ge- (25, 62). Future studies on cells and tis- •NO (84) and, presumably, peroxynitrite netically engineered cells of the enzymes sues from these patients may offer impor- Ϫ to NO2 . that participate in the formation or elimi- tant opportunities to understand the nation of nitrating species are available. mechanisms of biological protein nitration Lessons from Pharmacology The preferential nitration pathways under in humans. Pharmacological experiments have been various pathologically relevant conditions instrumental in defining the biological have been assessed by genetic approaches mechanisms of nitration. Inhibition of that involve SODs, namely overexpression NOS consistently leads to inhibition of of MnSOD or CuZnSOD or heterozygous **On the contrary, expression of amyotrophic lateral scle- Ϫ • ͞ MnSOD knockouts and MPO and EPO rosis-associated mutants of CuZnSOD leads to increased nitration, and the sources of O2 H2O2 motoneuron nitration and apoptosis (59), possibly due to can be discriminated with inhibitors of knockouts. MnSOD or CuZnSOD over- a toxic gain of function of SOD that promotes peroxyni- NAD(P)H oxidases, xanthine oxidase, or expressors decreased •NO-dependent in- trite formation and nitration reactions.

Radi PNAS ͉ March 23, 2004 ͉ vol. 101 ͉ no. 12 ͉ 4007 Downloaded by guest on September 28, 2021 Conclusion and Perspectives contribution of the peroxynitrite and matory, vascular, and neurodegenerative ͞ The analysis presented herein shows that hemeperoxidase transition metal-depen- diseases. biological protein tyrosine nitration†† dent pathways to biological nitration, the I thank Drs. Stanley L. Hazen, Harry Ischiro- mainly occurs through free radical path- detection of nitrated proteins with altered function in vivo, and the data arising from poulos, Bruce A. Freeman, Joe S. Beckman, ways, and that there is an interplay involv- Irwin Fridovich, Jonathan S. Stamler, Jean ing excess •NO, oxidants, and transition pharmacological and genetic approaches Claude Drapier, and Ronald P. Mason for metal centers. The individual or combined support the significance of nitration as a valuable comments, and my coinvestigators in biochemical process linked to •NO-depen- Montevideo for contributions to the elucida- dent pathophysiology. Approaches di- tion of various aspects presented in the rected at inhibiting the oxidative modifica- manuscript. This work was supported †† by the Howard Hughes Medical Institute, Biological nitration is not restricted only to tyrosine nitrations caused by reactive nitrogen species, tion but may also result in nitration of tryptophan resi- Fogarty–National Institutes of Health, the dues, as well as DNA bases, sugars, and lipids that can including protein tyrosine nitration, open John Simon Guggenheim Memorial Founda- result in altered or new biological activities. new avenues for the treatment of inflam- tion, and Universidad de la Repu´blica.

1. Ignarro, L. J. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 535–560. 37. Go, Y. M., Patel, R. P., Maland, M. C., Park, H., Beckman, J. S., 69. MacMillan-Crow, L. A., Crow, J. P., Kerby, J. D., Beckman, J. S. 2. Radi, R., Denicola, A., Alvarez, B., Ferrer-Sueta, G. & Rubbo, Darley-Usmar, V. M. & Jo, H. (1999) Am. J. Physiol. 277, & Thompson, J. A. (1996) Proc. Natl. Acad. Sci. USA 93, H. (2000) in Nitric Oxide, ed. Ignarro, L. (Academic, San Diego), H1647–H1653. 11853–11858. pp. 57–82. 38. Mihm, M. J., Wattanapitayakul, S. K., Piao, S. F., Hoyt, D. G. & 70. Castro, L., Eiserich, J. P., Sweeney, S., Radi, R. & Freeman, B. A. 3. Liu, X., Miller, M. J., Joshi, M. S., Thomas, D. D. & Lancaster, Bauer, J. A. (2003) Biochem. Pharmacol. 65, 1189–1197. (2004) Arch. Biochem. Biophys. 421, 99–107. J. R., Jr. (1998) Proc. Natl. Acad. Sci. USA 95, 2175–2179. 39. Cosentino, F., Eto, M., De Paolis, P., van der Loo, B., Bach- 71. Guo, W., Adachi, T., Matsui, R., Xu, S., Jiang, B., Zou, M. H., 4. Denicola, A., Batthyany, C., Lissi, E., Freeman, B. A., Rubbo, H. schmid, M., Ullrich, V., Kouroedov, A., Delli Gatti, C., Joch, H., Kirber, M., Lieberthal, W. & Cohen, R. A. (2003) Am. J. Physiol. & Radi, R. (2002) J. Biol. Chem. 277, 932–936. Volpe, M., et al. (2003) Circulation 107, 1017–1023. 285, H1396–H1403. 40. Augusto, O., Bonini, M. G., Amanso, A. M., Linares, E., Santos, 5. Sokolovsky, M., Riordan, J. F. & Vallee, B. L. (1966) Biochem- 72. Schmidt, P., Youhnovski, N., Daiber, A., Balan, A., Arsic, M., C. C. & De Menezes, S. L. (2002) Free Radical Biol. Med. 32, istry 5, 3582–3589. Bachschmid, M., Przybylski, M. & Ullrich, V. (2003) J. Biol. 841–859. 6. Beckman, J. S., Ischiropoulos, H., Zhu, L., van der Woerd, M., Chem. 278, 12813–12819. Smith, C., Chen, J., Harrison, J., Martin, J. C. & Tsai, M. (1992) 41. Hazen, S. L., Gaut, J. P., Hsu, F. F., Crowley, J. R., d’Avignon, A. & Heinecke, J. W. (1997) J. Biol. Chem. 272, 16990–16998. 73. Bachschmid, M., Thurau, S., Zou, M. H. & Ullrich, V. (2003) Arch. Biochem. Biophys. 298, 438–445. FASEB J. 17, 914–916. 7. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., 42. Fridovich, I. (1997) J. Biol. Chem. 272, 18515–18517. 265, 74. Stuehr, D., Pou, S. & Rosen, G. M. (2001) J. Biol. Chem. 276, Smith, C. D. & Beckman, J. S. (1992) Arch. Biochem. Biophys. 43. Yamazaki, I. & Piette, L. H. (1990) J. Biol. Chem. 13589– 13594. 14533–14536. 298, 431–437. 44. Burner, U., Furtmuller, P. G., Kettle, A. J., Koppenol, W. H. & 75. Shishehbor, M. H., Aviles, R. J., Brennan, M. L., Fu, X., 8. Ischiropoulos, H., Zhu, L. & Beckman, J. S. (1992) Arch. Obinger, C. (2000) J. Biol. Chem. 275, 20597–20601. Goormastic, M., Pearce, G. L., Gokce, N., Keaney, J. F., Jr., Biochem. Biophys. 298, 446–451. 45. Sampson, J. B., Rosen, H. & Beckman, J. S. (1996) Methods Penn, M. S., Sprecher, D. L., et al. (2003) J. Am. Med. Assoc. 289, 9. Beckmann, J. S., Ye, Y. Z., Anderson, P. G., Chen, J., Accavitti, Enzymol. 269, 210–218. 1675–1680. M. A., Tarpey, M. M. & White, C. R. (1994) Biol. Chem. 46. Ferrer-Sueta, G., Quijano, C., Alvarez, B. & Radi, R. (2002) 76. Brennan, M. L., Penn, M. S., Van Lente, F., Nambi, V., Hoppe–Seyler 375, 81–88. Methods Enzymol. 349, 23–37. Shishehbor, M. H., Aviles, R. J., Goormastic, M., Pepoy, M. L., 10. Ohshima, H., Friesen, M., Brouet, I. & Bartsch, H. (1990) Food 47. Bouton, C. & Drapier, J. C. (2003) Sci. STKE 182, pe17. McErlean, E. S., Topol, E. J., et al. (2003) N. Engl. J. Med. 349, Chem. Toxicol. 28, 647–652. 48. Soum, E., Brazzolotto, X., Goussias, C., Bouton, C., Moulis, 11. Pfeiffer, S., Schmidt, K. & Mayer, B. (2000) J. Biol. Chem. 275, 1595–1604. J. M., Mattioli, T. A. & Drapier, J. C. (2003) Biochemistry 42, 77. Radi, R., Cassina, A., Hodara, R., Quijano, C. & Castro, L. 6346–6352. 7648–7654. 12. Thomas, D. D., Espey, M. G., Vitek, M. P., Miranda, K. M. & (2002) Free Radical Biol. Med. 33, 1451–1464. 49. Gunther, M. R., Hsi, L. C., Curtis, J. F., Gierse, J. K., Marnett, 78. Turko, I. V., Li, L., Aulak, K. S., Stuehr, D. J., Chang, J. Y. & Wink, D. A. (2002) Proc. Natl. Acad. Sci. USA 99, 12691–12696. L. J., Eling, T. E. & Mason, R. P. (1997) J. Biol. Chem. 272, Murad, F. (2003) J. Biol. Chem. 278, 33972–33977. 13. Eiserich, J. P., Hristova, M., Cross, C. E., Jones, A. D., Freeman, 17086–17090. 79. Pearce, L. L., Pitt, B. R. & Peterson, J. (1999) J. Biol. Chem. 274, B. A., Halliwell, B. & van der Vliet, A. (1998) Nature 391, 50. Esteves, P. M., De M Carneiro, J. W., Cardoso, S. P., Barbosa, 35763–35767. 393–397. A. G., Laali, K. K., Rasul, G., Prakash, G. K. & Olah, G. A. 14. Bian, K., Gao, Z., Weisbrodt, N. & Murad, F. (2003) Proc. Natl. (2003) J. Am. Chem. Soc. 125, 4836–4849. 80. Pietraforte, D., Salzano, A. M., Scorza, G., Marino, G. & Acad. Sci. USA 100, 5712–5717. 51. Quijano, C., Hernandez-Saavedra, D., Castro, L., McCord, J. M., Minetti, M. (2001) Biochemistry 40, 15300–15309. 15. Brennan, M. L., Wu, W., Fu, X., Shen, Z., Song, W., Frost, H., Freeman, B. A. & Radi, R. (2001) J. Biol. Chem. 276, 11631– 81. Herold, S., Shivashankar, K. & Mehl, M. (2002) Biochemistry 41, Vadseth, C., Narine, L., Lenkiewicz, E., Borchers, M. T., et al. 11638. 13460–13472. (2002) J. Biol. Chem. 277, 17415–17427. 52. Whiteman, M., Siau, J. L. & Halliwell, B. (2003) J. Biol. Chem. 82. Romero, N., Radi, R., Linares, E., Augusto, O., Detweiler, C. D., 16. Fukuto, J. & Ignarro, L. (1997) Acc. Chem. Res. 30, 149–152. 278, 8380–8384. Mason, R. P. & Denicola, A. (2003) J. Biol. Chem. 278, 44049– 17. Eu, J. P., Liu, L., Zeng, M. & Stamler, J. S. (2000) Biochemistry 53. Aslan, M., Ryan, T. M., Townes, T. M., Coward, L., Kirk, M. C., 44057. 39, 1040–1047. Barnes, S., Alexander, C. B., Rosenfeld, S. S. & Freeman, B. A. 83. Romero, N., Denicola, A., Souza, J. M. & Radi, R. (1999) Arch. 18. Denicola, A., Souza, J. M. & Radi, R. (1998) Proc. Natl. Acad. (2003) J. Biol. Chem. 278, 4194–4204. Biochem. Biophys. 368, 23–30. Sci. USA 95, 3566–3571. 54 Grune, T., Blasig, I. E., Sitte, N., Roloff, B., Haseloff, R. & 84. Cosby, K., Partovi, K. S., Crawford, J. H., Patel, R. P., Reiter, 19. Radi, R., Peluffo, G., Alvarez, M. N., Naviliat, M. & Cayota, A. Davies, K. J. (1998) J. Biol. Chem. 273, 10857–10862. C. D., Martyr, S., Yang, B. K., Waclawiw, M. A., Zalos, G., Xu, (2001) Free Radical Biol. Med. 30, 463–488. 55 Irie, Y., Saeki, M., Kamisaki, Y., Martin, E. & Murad, F. (2003) X., et al. (2003) Nat. Med. 9, 1498–1505. 20. Gryglewski, R. J., Palmer, R. M. & Moncada, S. (1986) Nature Proc. Natl. Acad. Sci. USA 100, 5634–5639. 85. Estevez, A. G., Sampson, J. B., Zhuang, Y., Spear, N., Richard- 320, 454–456. 56. Rubbo, H., Radi, R., Trujillo, M., Telleri, R., Kalyanaraman, B., son, G. J., Crow, J. P., Tarpey, M. M., Barbeito, L. & Beckman, 21. Rubanyi, G. M. & Vanhoutte, P. M. (1986) Am. J. Physiol. 250, Barnes, S., Kirk, M. & Freeman, B. A. (1994) J. Biol. Chem. 269, J. S. (2000) Free Radical Biol. Med. 28, 437–446. H822–H827. 26066–26075. 86. Bartesaghi, S., Trujillo, M., Denicola, A., Folkes, L., Wardman, 57. Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., 22. Ignarro, L. J., Byrns, R. E., Buga, G. M., Wood, K. S. & P. & Radi, R. (2004) Free Radical Biol. Med. 36, 471–483. Wink, D. A. & Grisham, M. B. (1996) J. Biol. Chem. 271, 40–47. Chaudhuri, G. (1988) J. Pharmacol. Exp. Ther. 244, 181–189. 87. Scott, G. S. & Hooper, D. C. (2001) Med. Hypotheses 56, 95–100. 58. Goldstein, S., Czapski, G., Lind, J. & Merenyi, G. (2000) J. Biol. 23. Liochev, S. I. & Fridovich, I. (2002) Free Radical Biol. Med. 33, 88. Trujillo, M., Alvarez, M. N., Peluffo, G., Freeman, B. A. & Radi, 137–141. Chem. 275, 3031–3036. 59. Estevez, A. G., Crow, J. P., Sampson, J. B., Reiter, C., Zhuang, R. (1998) J. Biol. Chem. 273, 7828–7834. 24. Buerk, D. G., Lamkin-Kennard, K. & Jaron, D. (2003) Free 89. Ferrer-Sueta, G., Vitturi, D., Batinic-Haberle, I., Fridovich, I., Radical Biol. Med. 34, 1488–1503. Y., Richardson, G. J., Tarpey, M. M., Barbeito, L. & Beckman, J. S. (1999) Science 286, 2498–2500. Goldstein, S., Czapski, G. & Radi, R. (2003) J. Biol. Chem. 278, 25. Clark, S. R., Coffey, M. J., Maclean, R. M., Collins, P. W., Lewis, 27432–27438. M. J., Cross, A. R. & O’Donnell, V. B. (2002) J. Immunol. 169, 60. Fries, D. M., Paxinou, E., Themistocleous, M., Swanberg, E., Griendling, K. K., Salvemini, D., Slot, J. W., Heijnen, H. F., 90. Muscoli, C., Cuzzocrea, S., Riley, D. P., Zweier, J. L., Thiemer- 5889–5896. Hazen, S. L. & Ischiropoulos, H. (2003) J. Biol. Chem. 278, mann, C., Wang, Z. Q. & Salvemini, D. (2003) Br. J. Pharmacol. 26 Murphy, M. E. & Sies, H. (1991) Proc. Natl. Acad. Sci. USA 88, 22901–22907. 140, 445–460. 10860–10864. 61. Jiang, Q. & Hurst, J. K. (1997) J. Biol. Chem. 272, 32767–32772. 91. Pacher, P., Liaudet, L., Bai, P., Mabley, J. G., Kaminski, P. M., 27 Jourd’heuil D., Laroux, F. S., Miles, A. M., Wink, D. A. & 62. Rosen, H., Crowley, J. R. & Heinecke, J. W. (2002) J. Biol. Chem. Virag, L., Deb, A., Szabo, E., Ungvari, Z., Wolin, M. S., Groves, Grisham, M. B. (1999) Arch. Biochem. Biophys. 361, 323–330. 277, 30463–30468. J. T. & Szabo, C. (2003) Circulation 107, 896–904. 28. Hobbs, A. J., Fukuto, J. M. & Ignarro, L. (1994) Proc. Natl. Acad. 63. Baldus, S., Eiserich, J. P., Brennan, M. L., Jackson, R. M., 91, 92. Keller, J. N., Kindy, M. S., Holtsberg, F. W., St Clair, D. K., Yen, Sci. USA 10992–10996. Alexander, C. B. & Freeman, B. A. (2002) Free Radical Biol. 29. Sowers, J. R. (2002) N. Engl. J. Med. 346, 1999–2001. H. C., Germeyer, A., Steiner, S. M., Bruce-Keller, A. J., Med. 33, 1010. Hutchins, J. B. & Mattson, M. P. (1998) J. Neurosci. 18, 687–697. 30. Gutierrez, H. H., Nieves, B., Chumley, P., Rivera, A. & Freeman, 64. Hazen, S. L., Zhang, R., Shen, Z., Wu, W., Podrez, E. A., B. A. (1996) Free Radical Biol. Med. 21, 43–52. 93. Wang, H. D., Johns, D. G., Xu, S. & Cohen, R. A. (2002) Am. J. MacPherson, J. C., Schmitt, D., Mitra, S. N., Mukhopadhyay, C., Physiol. 282, H1697–H1702. 31. Sarti, P., Avigliano, L., Gorlach, A. & Brune, B. (2002) Cell et al. Circ. Res. 85, Chen, Y., (1999) 950–958. 94. Kim, G. W. & Chan, P. H. J (2002) Cereb. Blood Flow Metab. 22, Death Differ. 9, 1160–1162. 65. Souza, J. M., Daikhin, E., Yudkoff, M., Raman, C. S. & 798–809. 32. Radi, R., Beckman, J. S., Bush, K. M. & Freeman, B. A. (1991) Ischiropoulos, H. (1999) Arch. Biochem. Biophys. 371, 169–178. 95. Gaut, J. P., Byun, J., Tran, H. D., Lauber, W. M., Carroll, J. A., J. Biol. Chem. 266, 4244–4250. 66. Cassina, A., Hodara, R., Souza, J., Thomson, L., Castro, L., J. Clin. 33. Trujillo, M. & Radi, R. (2002) Arch. Biochem. Biophys. 397, Ischiropoulos, H., Freeman, B. A. & Radi, R. (2000) J. Biol. Hotchkiss, R. S., Belaaouaj, A. & Heinecke, J. W. (2002) 91–98. Chem. 275, 21409–21415. Invest. 109, 1311–1319. 34. Arteel, G. E., Briviba, K. & Sies, H. (1999) FEBS Lett. 445, 67. Vadseth, C., Souza, J. M., Thomson, L., Seagraves, A., Na- 96. Brennan, M. L. Anderson, M. M., Shih, D. M., Qu, X. D., Wang, 226–230. gaswami, C., Scheiner, T., Torbet, J., Vilaire, G., Bennett, J. S., X., Mehta, A. C., Lim, L. L., Shi, W., Hazen, S. L., Jacob, J. S., 35. Bryk, R., Griffin, P. & Nathan, C. (2000) Nature 407, 211–215. Murciano, J. C., et al. (2003) J. Biol. Chem., jbc͞M306101200v1. et al. (2001) J. Clin. Invest. 107, 419–430. 36. Brito, C., Naviliat, M., Tiscornia, A. C., Vuillier, F., Gualco, G., 68. Balafanova, Z., Bolli, R., Zhang, J., Zheng, Y., Pass, J. M., 97. LaVaute, T., Smith, S., Cooperman, S., Iwai, K., Land, W., Dighiero, G., Radi, R. & Cayota, A. M. (1999) J. Immunol. 162, Bhatnagar, A., Tang, X.-L., Wang, O., Cardwell, E. & Ping, P. Meyron-Holtz, E., Drake, S. K., Miller, G., Abu-Asab, M., 3356–3366. (2002) J. Biol. Chem. 277, 15021–15027. Tsokos, M., et al. (2001) Nat. Genet. 27, 209–214.

4008 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307446101 Radi Downloaded by guest on September 28, 2021