Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide

Free Radical Biology & Medicine 42 (2007) 1359 – 1368 www.elsevier.com/locate/freeradbiomed

Original Contribution

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide Verónica Demicheli a,b , Celia Quijano a,b , Beatriz Alvarez b,c , Rafael Radi a,b,⁎ a

b

Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. Gral. Flores 2125, 11800 Montevideo, Uruguay Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Avda. Gral. Flores 2125, 11800 Montevideo, Uruguay c Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay Received 24 July 2006; revised 22 December 2006; accepted 21 January 2007 Available online 24 January 2007

Abstract Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO2U−) and nitric oxide (JUNO). The inactivation was also observed with varying JUNO/JO2U− ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to JUNO and JO2U− in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant JUNO and JO2U−. The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metalcatalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a UNO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions. © 2007 Elsevier Inc. All rights reserved. Keywords: MnSOD; CuZnSOD; Peroxynitrite; Nitric oxide; Superoxide; Free radical; Tyrosine nitration

Introduction

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Superoxide radical (O2 −) is formed in vivo through the oneelectron reduction of molecular oxygen by flavoproteins like xanthine oxidase [1,2], the mitochondrial respiratory chain [3], activation of NADPH oxidase [4,5], and oxidation of quinols [6]. Under normal conditions, superoxide formation rates are assumed to be as high as 34 μM/min in mitochondria [3,7]. However, intracellular superoxide steady-state concentrations are kept very low (∼10−10 M) [3,7,8] due to the action of superoxide

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Abbreviations: BSA, bovine serum albumin; DMPO, 5,5-dimethyl-1pyrroline N-oxide; NBT, 4-nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase; TBS, Tris saline buffer. ⁎ Corresponding author. Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800 Montevideo, Uruguay. Fax: +598 2 9249563. E-mail address: [email protected] (R. Radi). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.01.034

dismutases (SOD), CuZnSOD and MnSOD, which catalyze the dismutation of superoxide to hydrogen peroxide and molecular oxygen with rate constants of 1–2 × 109 M−1 s−1 [9–11]. Despite its free radical nature, superoxide is in general not too reactive; its principal targets are [4Fe–4S]-containing dehydratases, such as aconitase [8,12]. However, superoxide can react with nitric oxide ( NO), an inorganic radical that is also formed in vivo by the action of nitric oxide synthase (NOS), at nearly diffusion-limited rates (0.5–1.9 × 1010 M−1 s−1) to form peroxynitrite [13–15]. Peroxynitrite 1 is a potent one- and two-electron oxidant (E°′ (ONOOH, H+/ NO2, H2O) = 1.6–1.7 V and E°′(ONOOH, H+/ NO2−, H2O) = 1.3–1.37 V) [16,17] and a nitrating species. This molecule can react directly with several targets such as thiols [18], protein metal centers [19], carbon dioxide [20,21], and

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1 The term peroxynitrite is used to refer to both peroxynitrite anion and peroxynitrous acid. IUPAC-recommended names are oxoperoxonitrate (1-) and hydrogen oxoperoxonitrate, respectively.

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selenium compounds [22]. At pH 7.4, peroxynitrite is mostly in its anionic form but in equilibrium with its conjugated acid, peroxynitrous acid (pKA6.8) which can then homolyze at a rate of 0.9 s−1 at pH 7.4, 37°C [18,23], forming nitrogen dioxide ( NO2) and hydroxyl radical ( OH) in a ∼30% yield [24,25]. These radicals participate in peroxynitritedependent toxicity, being involved in tyrosine nitration [26,27] and lipid peroxidation [28,29] processes. Nitration and oxidation of free tyrosine by simultaneous fluxes of nitric oxide and superoxide have been related to peroxynitrite formation by several authors [30–34]. In one of those studies [30], the maximum yield of nitration was achieved at equal rates of superoxide and nitric oxide formation, generating a “bell-shaped” curve in which at high nitric oxide/ superoxide or superoxide/nitric oxide ratios, tyrosine nitration was inhibited. In recent theoretical reports, including one of our group [35,36], this bell-shaped profile was completely lost, due to the incorporation of biologically relevant events such as nitric oxide diffusion and superoxide-catalyzed dismutation. Moreover, nitration became responsive to increases in nitric oxide and superoxide rates, irrespective of the relative superoxide/ nitric oxide flux ratio and in agreement with in vivo observations (for a critical analysis see [27]). Under physiological conditions most of the superoxide produced in the cell dismutates in the reaction catalyzed by SOD [37] and only marginal basal levels of tyrosine nitration are observed [38]. However, under pathological conditions when nitric oxide levels increase due to the expression of inducible NOS (iNOS) or overactivation of endothelial NOS (eNOS) and neuronal NOS (nNOS) [39], biological markers of ONOO− formation, such as 3-nitrotyrosine are substantially augmented [38,40–42]. Thus, nitric oxide reaction with superoxide occurs despite the SOD-catalyzed dismutation of superoxide posing a “kinetic dilemma” whose resolution can be aided by specific in vitro and in silico approaches. Moreover, experimental evidence in vivo indicates that MnSOD is nitrated and inactivated under pathological conditions [38,41,43–46], with nitration at the active site tyrosine-34 being mainly responsible for the loss of activity [38,47,48]. Both MnSOD and CuZnSOD react directly with peroxynitrite with second-order rate constants of 2.5 × 104 and 9.4 × 103 M−1 s−1 per monomer, respectively [49,50]. Both enzymes are, also, inactivated by peroxynitrite [47,50,51] with tyrosine nitration the main modification observed in MnSOD, which has three tyrosine residues susceptible to nitration (Y-45, Y-193, and the critical for activity Y-34) [48]. In CuZnSOD histidinyl radical formation was observed after exposure to peroxynitrite [50] while tyrosine nitration cannot occur due to the lack of this residue in the primary structure of this enzyme. In both cases, a direct reaction between peroxynitrite and the metal centers of the enzymes is proposed as mainly responsible for their sitespecific inactivation and, as for other Lewis acids, it has been proposed that peroxynitrite reacts with the metal center forming an adduct that homolyzes to yield nitrogen dioxide and the corresponding oxyradical [49,50]. However, it is still not clear, in the case of MnSOD, how nitrogen dioxide arising from the reaction of peroxynitrite with manganese ions can lead to

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tyrosine nitration in the presence of biologically relevant levels of glutathione (5–10 mM) [36] which readily (k = 2 × 107 M−1 s−1) scavenges this oxidant [52]. Herein, we exposed human superoxide dismutases (CuZnSOD and MnSOD) to simultaneous fluxes of nitric oxide and superoxide, and studied their inactivation along with the formation of protein radicals and 3-nitrotyrosine. This approach provides a way to prove that biological fluxes of nitric oxide can outcompete superoxide from the enzymatic dismutation to form peroxynitrite, in a system that more closely resembles a biological condition. Moreover, the occurrence of nitration in the presence of relevant levels of glutathione was investigated. Materials and methods Chemical reagents Spermine NONOate was obtained from Alexis, xanthine oxidase from Calbiochem, and catalase from Fluka. Doubledistilled 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was generously provided by Dr. R. Mason (NIEHS, NC). Hydrogen peroxide was from J. T. Baker and its concentration was determined from the absorbance at 240 nm (ε = 43.6 M−1 cm−1) [53]. All other reagents were from Sigma. Peroxynitrite solutions were prepared from acidified hydrogen peroxide and sodium nitrite as described [54]. Hydrogen peroxide contamination was eliminated by the addition of manganese dioxide and peroxynitrite concentration was determined measuring the absorbance at 302 nm (ε302 = 1.67 mM−1 cm−1). The concentration of nitrite present as contaminant was measured with the Griess reagent [55,56] after decay of peroxynitrite to nitrate in monobasic sodium phosphate solution and was less than 30% of peroxynitrite. Human recombinant MnSOD was a kind gift from Dr. Daniel Hernández and Dr. Joseph McCord (WebbWaring Institute, University of Colorado, Denver, CO) [49]. Human recombinant CuZnSOD purification The enzyme was purified as described previously [50]. Briefly, the human CuZnSOD protein was expressed using the Escherichia coli strain BL21(DE3)plys S, transformed with a pET plasmid encoding wild-type human recombinant CuZnSOD (kind gift from Dr. Joseph Beckman, State University, Corvallis, OR). Cultures were grown in Luria Broth containing ampicillin and chloramfenicol at 37°C. CuZnSOD expression was induced by adding 1 mM isopropyl β-D-thiogalactopyranoside. After 4 h at 24°C, cells were harvested by centrifugation, resuspended in 20 mM Tris-HCl, pH 7.8, and frozen overnight. The suspension was then thawed, sonicated, treated with DNAase, and centrifuged for 15 min at 18,000g. After that, ZnCl2 (1 mM) and CuCl2 (1 mM) metal salts were added, and a remarkable change in the color of the extract was observed. CuZnSOD was then purified through ammonium sulfate fractionation followed by ion-exchange chromatography in Sepharose CL6B and gel filtration in Sephadex G-75. The protein concentration was determined through the enhanced BCA assay (Pierce) using bovine CuZnSOD or

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albumin as standard. Human recombinant CuZnSOD and MnSOD had specific activities of 3.5 × 103 and 2.9 × 103 U mg−1, respectively. Superoxide dismutase activity The dismutase activity was measured by the decrease in the rate of superoxide-dependent cytochrome c reduction at 550 nm using xanthine/xanthine oxidase as superoxide source [57,58]. Activity was also assessed in native 15% polyacrilamide gels stained with 4-nitroblue tetrazolium (NBT) [58]. The bands were revealed through the reduction of NBT (0.25 mg/mL) by superoxide produced by the photochemical reduction of riboflavin (0.1 mg/mL) with N,N,N′,N′-Tetramethyl-ethylenediamine (TEMED, 1%). Determination of superoxide and nitric oxide fluxes Nitric oxide release from spermine NONOate (half-life of 39 min at 37°C) was determined measuring the oxyhemoglobin oxidation rate at 577 nm (ε577 = 0.011 μM−1 cm−1 [59]) or monitoring the donor decay at 252 nm (ε252 = 8.5 mM−1 cm−1 [60]). Superoxide was generated using xanthine oxidase plus hypoxanthine (2 mM) and its release was determined by monitoring the SOD-inhibitable cytochrome c reduction at 550 nm. Flux rates of superoxide were calculated using an extinction coefficient of 21 mM−1 cm−1 [61] and measured before each experiment. Both fluxes of nitric oxide (J NO) and superoxide (JO2 −) were constant within the times of the experiments.

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Exposure of SOD to nitric oxide and superoxide fluxes Nitric oxide and superoxide generating systems were incubated at the stated concentrations in the presence of CuZnSOD or MnSOD for the indicated times at 37°C, in 0.1 M phosphate buffer, pH 7.4, with 0.1 mM DTPA. Reactions were stopped by gel filtration with MicroBiospin 6 chromatography columns (BioRad) or by immediate dilution (15-fold) and cooling after the exposure. The levels of oxygen in the system were carefully controlled using an YSI 5300 biological oxygen monitor (YSI Inc.), and the incubation periods were defined in order to have enough oxygen to fully support the xanthine oxidase-catalyzed reaction and a linear production of superoxide. As a control oxygen consumption of xanthine oxidase was measured in the presence of spermineNONOate, in order to assess if the enzyme was inactivated by the products of the reaction. In agreement with previous reports [62], the enzyme activity was not affected at the measured time (10 min). At longer times of exposure, the reaction mixture was gently bubbled with oxygen to recover oxygen saturation levels and aliquots of fresh xanthine oxidase were added every 5–10 min if superoxide formation decrease, detected by oxygen consumption, was observed. Western blot analyses Proteins were subjected to 13% SDS-polyacrylamide gel electrophoresis, transferred overnight to nitrocellulose mem-

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branes and blocked with 5% bovine serum albumin (BSA), 0.6% Tween 20 in 50 mM Tris saline buffer (TBS) (blocking buffer) for 1 h. Membranes were then probed with either rabbit polyclonal anti-nitrotyrosine antibody (1/2000 dilution) (raised in our laboratory [63]), sheep polyclonal anti-human MnSOD (1/1000 dilution) (Calbiochem), and rabbit polyclonal anti-human CuZnSOD (1/200 dilution), raised against human recombinant CuZnSOD (kind gift of Mónica Marín, Universidad de la República, Uruguay) primary antibodies. For the detection, immunocomplexed membranes were incubated 1 h in blocking buffer, washed in TBS containing 0.3% Tween 20 (washing buffer), and probed for 1 h with anti-rabbit IgG (CuZnSOD and nitrotyrosine detection) or anti-sheep IgG (MnSOD detection) horseradish peroxidaselinked secondary antibody (1/10,000 dilution) (Calbiochem) in blocking buffer. After extensive washing of the probed membranes, immunoreactive proteins were visualized with a luminol-enhanced chemiluminescence detection kit (ECL) (Bio-Rad). Immuno-spin trapping analyses After the exposure of CuZnSOD and MnSOD to simultaneous fluxes of nitric oxide and superoxide in the presence of 100 mM DMPO, the samples were transferred to nitrocellulose membranes from SDS-polyacrylamide gels (under the conditions described earlier). Membranes were blocked with 2.5% BSA, 2.5% milk, and 0.3% Tween 20 in TBS for 1 h and further exposed to the rabbit polyclonal anti-DMPO nitrone primary antibody (rabbit anti-DMPO serum, 1/5000 dilution, kind gift from Dr. R. Mason, NIEHS, NC) for 1 h [64]. After extensive washing with 0.1% BSA, 0.1% milk, and 0.3% Tween 20 in TBS, membranes were probed with anti-rabbit IgG horseradish peroxidase-linked secondary antibody and the immunoreactive proteins were visualized by ECL under the same conditions described above [65]. Simulations Computer-assisted simulations, using reactions and rate constants reported in the literature, were performed with the software Gepasi (version 3.3) [66,67]. Data analysis Results are expressed as means ± SD unless otherwise specified or by a representative example. Results CuZnSOD and MnSOD are inactivated by fluxes of nitric oxide and superoxide Exposure of CuZnSOD (5 μM, dimer) and MnSOD (5 μM, tetramer) to simultaneous equimolar fluxes of nitric oxide (J NO) and superoxide (JO2 −) (10 μM/min) led to a timedependent inactivation of both enzymes (Fig. 1A). Total

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superoxide. Thus, since both CuZnSOD and MnSOD are reported to be inactivated by bolus addition of peroxynitrite, our data are consistent with peroxynitrite formation in the studied system. Moreover, exposure of CuZnSOD to different rates of equimolar J NO and JO2 − (10 μM/min for 10 min or 30 μM/min for 3 min) led to similar values of inactivation, in agreement with similar amounts of peroxynitrite being formed in these systems, which are responsible for the inactivation of the enzyme (Fig. 1B). In the case of MnSOD, tyrosine nitration was observed in the samples exposed to both fluxes, which increased as a function of time (Fig. 1C). This observation further supports peroxynitrite formation in our system. Immuno-spin trapping experiments using DMPO as a spin trap indicated that, simultaneously with inactivation, a protein radical was formed during the exposure of CuZnSOD and MnSOD (5 μM) to equimolar fluxes of nitric oxide and superoxide (Fig. 2). A similar result was observed when the enzymes (5 μM) were exposed to peroxynitrite (1 and 0.1 mM for CuZnSOD and MnSOD, respectively). In the case of CuZnSOD, the detection of a protein radical is in accordance to a previous report where a protein radical signal was detected by EPR after exposure to authentic peroxynitrite and attributed to histidinyl radical [50]. Based on the results, we could infer the formation of histidinyl radical in our system, an amino acid-derived radical susceptible to immuno-spin trapping as recently reported [70]. In MnSOD, the DMPO-protein signal may be due to the formation of tyrosyl radical, since, as it can be observed in Fig. 2B, the signal of protein 3nitrotyrosine detected by immunoblot decreased in the samples incubated with DMPO prior to the exposure to J NO and JO2 − or authentic peroxynitrite.

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Fig. 1. Time-dependent inactivation of MnSOD and CuZnSOD. (A) CuZnSOD (■) and MnSOD (●), 5 μM, were exposed to equimolar fluxes of superoxide and nitric oxide 10 μM/min, respectively for different times at 37°C, pH 7.4. (B) Native PAGE of CuZnSOD, stained with NBT. Superoxide dismutase activity is visible as colorless spots. CuZnSOD (5 μM) was exposed to equimolar JUNO and JO2U of 10 μM/min for 10 min and 30 μM/min for 3 min. SOD activity was measured using the superoxide-dependent cytochrome c reduction method. (C) Immunodetection of protein 3-nitrotyrosine in samples of MnSOD (5 μM) exposed to equimolar JUNO and JO2U− (10 μM/min) for 0, 3, 5, 10, 20, and 30 min.

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inactivation was not achieved during the times of exposure that were probed for both enzymes, with a maximum inactivation of ∼ 60% being achieved at 30 min of incubation. The exposure to only one of the fluxes (either superoxide or nitric oxide) did not affect the activity of the enzymes (Table 1) and fluxes of nitric oxide alone did not cause nitration (not shown). As hydrogen peroxide has been widely reported as an inhibitor of CuZnSOD activity [68,69] we added catalase (100 U/mL measured at 10 mM hydrogen peroxide) or hydrogen peroxide (50 μM) to the reaction mixture but neither one had an effect on the inactivation of CuZnSOD or MnSOD (Table 1). Therefore, hydrogen peroxide does not seem to be involved in the inactivation of the enzymes by fluxes of nitric oxide and Table 1 Activity of MnSOD and CuZnSOD (5 μM) exposed to fluxes of nitric oxide and superoxide (10 μM/min, 10 min) assessing the effect of uricase (2 U/mL), catalase (100 U/mL), and hydrogen peroxide (50 μM) Condition

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Control (SOD) SOD + J NO and JO2 − SOD + J NO SOD + JO2 − SOD + J NO and JO2 − + uricase SOD + J NO and JO2 − + catalase SOD + J NO and JO2 −+ H2O2

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CuZnSOD, % activity

MnSOD, % activity

100 ± 7 66 ± 13 99 ± 11 92 ± 8 78 ± 3 77 ± 12 72 ± 15

100 ± 5 68 ± 14 94 ± 2 99 ± 6 52 ± 4 58 ± 5 63 ± 9

Fig. 2. Detection of protein free radical formation in CuZnSOD and MnSOD exposed to nitric oxide and superoxide fluxes. (A) Immuno-spin trapping blot of CuZnSOD (5 μM) exposed to equimolar JUNO and JO2U− (10 μM/min for 10 min or 30 μM/min for 3 min) or peroxynitrite (1 mM) in the presence of DMPO (100 mM), detected using an anti-DMPO antibody. ROA stands for “reverseorder addition” (B) Immuno-spin trapping (upper panel) and immunodetection of protein 3-nitrotyrosine (lower panel) of MnSOD (5 μM) exposed to equimolar JUNO and JO2U− (10 μM/min, 10 min) and peroxynitrite (100 μM) in the presence and absence of DMPO (100 mM).

V. Demicheli et al. / Free Radical Biology & Medicine 42 (2007) 1359–1368

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Formation of covalent dimers was not detected for either MnSOD or CuZnSOD after the exposure to simultaneous J NO and JO2 − (10 μM/min) (not shown).

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Computer-assisted determination of superoxide, nitric oxide, and peroxynitrite steady-state concentrations In order to kinetically substantiate how peroxynitrite is being formed from superoxide and nitric oxide in the presence of SOD we performed computer-assisted simulations using the reactions and kinetic constants present in Table 2, along with J NO and JO2 − of 10 μM/min and SOD concentration (5 μM) as that present in our experiments. As shown in Fig. 3, the levels of nitric oxide and superoxide were dramatically affected by the presence of SOD. Superoxide levels, specifically, changed from nanomolar to picomolar concentrations after the addition of SOD (Figs. 3A and B). In contrast, the scavenging of superoxide by SOD raised the nitric oxide concentration from nanomolar to micromolar (Figs. 3C and D) which then could outcompete SOD reactions with superoxide and generate peroxynitrite. Thus, and remarkably, peroxynitrite steady-state concentration only decreased ∼ 37% in the presence of SOD (Figs. 3E and F), reaching a flux of formation of ∼ 5 μM/min. Therefore, peroxynitrite can be formed by superoxide and nitric oxide despite catalytic dismutation of superoxide and is then capable of reacting with multiple targets, including SOD itself.

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Table 2 Reactions that take place when nitric oxide and superoxide are generated simultaneously in the presence of SOD

1 2 3 4 5 6 7a 7b 8 9 10 11 13 14

Reaction

k

Reference

O2 − + NO → ONOO− O2 − + SOD → O2 + SODred SODred + O2 − + 2 H+ → SOD + H2O2 O2 − + O2 − + 2 H+ → H2O2 + O2 ONOO− → NO−3 ONOO−→ NO2 + OH CuZnSOD + ONOO− → CuZnSODinactive MnSOD + ONOO− → MnSODinactive OH + NO−2 → OH− + NO2 OH + NO → NO−2 + H+ OH + NO2 → ONOO− + H+ OH + O2 − → O2 + OH− 2 NO + O2 → 2 NO2 NO + NO2 ⇌ N2O3

5 × 109 M−1 s−1 2.0 × 109 M−1 s−1 2.0 × 109 M−1 s−1 2.5 × 105 M−1 s−1 0.63 s−1 0.27 s−1 9.1 × 103 M−1 s−1 1.0 × 105 M−1 s−1 1.0 × 1010 M−1 s−1 2.0 × 1010 M−1 s−1 4.5 × 109 M−1 s−1 7 × 109 M−1 s−1 2.9 × 106 M−2 s−1 1.1 × 109 M−1 s−1 (f ) a 8.4 × 104 s−1 (r) 4.5 × 108 M−1 s−1 (f ) 6.9 × 103 s−1 (r) 8.2 × 104 s−1 b 1 × 103 s−1 4.5 × 109 M−1 s−1 (f ) 1.1 s−1 (r) 1.3 s−1 3.1 × 108 M−1s−1 4.8 × 109 M−1s−1

[14] [9] [9] [79] [80] [80] [50] [49] [75] [75] [82] [75] [82] [75]

U U U U U U U

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U U U U U U U U U U U U U 15 2 NO ⇌ N O 2

2

4

16 N2O3 + H2O → 2 NO−2 + 2H+ 17 N2O4 + H2O → NO−2 + NO−3 + 2H+ 18 NO2 + O2 − ⇌ O2NOO−

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19 O2NOO− → NO−2 +O2 20 N2O3 + ONOO− → NO−2 + 2 NO2 21 ONOO− + OH → ONOO + OH−

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a b

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[75] [82] [30] [83] [83] [84] [81]

(f) and (r) represent forward and reverse kinetic constant, respectively. Calculated from the following equation: k = 2 × 103 + 8 × 105 [phosphate] (s−1).

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SOD inactivation by fluxes of nitric oxide and superoxide is partially inhibited by glutathione but not by uric acid Uric acid is a known scavenger of nitrogen dioxide and hydroxyl radical derived from peroxynitrite homolysis [31,32,71]. However, in our system, the addition of uric acid at concentrations of 100 μM and 1 mM (the latter was only evaluated in MnSOD) did not protect the enzymes from inactivation by simultaneous J NO and JO2 − (10 μM/min) (Fig. 4A), although it did decrease, albeit not completely, tyrosine nitration in MnSOD (Fig. 4B). Nitration of MnSOD by bolus addition of peroxynitrite was also diminished in the presence of uric acid (Fig. 4B), while inactivation was not affected (not shown). Also, the addition of uricase did not enhance significantly the inactivation levels of SOD by the fluxes (Table 1). Importantly, while glutathione (1 and 10 mM), known to react with both peroxynitrite (k = 1.35 × 103 M−1s−1 [23]) and nitrogen dioxide [52], caused total protection from MnSOD inactivation (not shown) and nitration (Fig. 4D) on addition of authentic peroxynitrite in bolus addition (100 μM) or infusion (10 μM/min, 10 min), it was only partially inhibitory (ca. 75–80% protection) on both processes in the presence of the more biologically relevant J NO and JO2 − (Figs. 4C and D). These results with uric acid and glutathione suggest that (a) the inactivation of CuZn- and MnSOD is due to the direct reaction of peroxynitrite with the enzymes and not the radicals derived from peroxynitrite homolysis and (b) in the case of MnSOD, a nitrogen dioxide-independent pathway of nitration may be involved [27,72].

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SOD inactivation persists at variable nitric oxide/superoxide flux ratios

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When SODs were exposed to a constant JO2 − (10 or 30 μM/ min) and varying J NO, we observed that at higher nitric oxide fluxes the enzyme was persistently inactivated with a maximum inactivation, in CuZnSOD (5 μM), of 50% at a 3/1 J NO/JO2 − (Fig. 5A), where the steady state of peroxynitrite reaches a plateau (Fig. 5A, inset). At high JO2 −/J NO (3/1), similar inactivation values were observed (not shown). A similar behavior was observed when exposing MnSOD (5 μM) to increasing J NO/JO2 −, achieving a maximum inactivation of ∼40% (Fig. 5B). Tyrosine nitration was observed under these conditions, increasing at higher J NO/JO2 − (Fig. 5B, inset).

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Discussion SOD inactivation by peroxynitrite in vitro is an extensively reported event [41,47–51], and firm evidence exists for the occurrence of the reaction in vivo for MnSOD [38,43–46]. Yet, the actual formation of peroxynitrite and its reaction with SOD are rather counterintuitive concepts, because superoxide reacts with the enzyme at close to diffusion-limited rates and SOD concentration is relatively high (4–40 μM CuZnSOD and 1– 30 μM MnSOD [49,73]). However, nitric oxide could potentially react with superoxide at even faster rates (k = 0.5– 1.9 × 1010 M−1 s−1 [13–15]), depending on production rates

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Fig. 3. Computer-assisted simulations of superoxide, nitric oxide and peroxynitrite concentration under the experimental conditions.The simulations were performed considering the conditions of the experiments. Simultaneous generation of 10 μM/min superoxide and nitric oxide for 10 min, in either the absence (left panels) or the presence (right panels) of CuZnSOD (5 μM), using the reactions presented in Table 2. Concentrations of superoxide (A and B), nitric oxide (C and D), and peroxynitrite (E and F) are shown.

and steady-state concentrations which remain largely undefined. Indeed, as we report herein, SOD is inactivated by simultaneous fluxes of superoxide and nitric oxide generated in vitro in agreement with peroxynitrite formation and subsequent reaction with SOD. The kinetic bases of these processes were

confirmed by computer-assisted simulations which indicated the formation of peroxynitrite at levels capable of reacting and inactivating SOD. Peroxynitrite, derived from the fluxes of superoxide and nitric oxide, inactivated both Cu,Zn- and MnSOD in a time-

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oxidative process). However, extent of inactivation by the “cumulative” dose of peroxynitrite (Fig. 1A) correlated well with that previously reported at similar SOD concentrations using authentic peroxynitrite [50]. Our results suggest that the radicals derived from the homolysis of peroxynitrite are not responsible for the inactivation of the enzymes because the catalytic removal of xanthine oxidase-generated uric acid, a scavenger of the radicals derived from peroxynitrite [71], by uricase did not enhance the inactivation (Table 1). Uric acid slowly reacts with peroxynitrite (k = 4.8 × 102 M−1 s−1) [74]; yet at the concentrations used in our assay (100 μM and 1 mM) this reaction is outcompeted by peroxynitrite reaction with MnSOD and the proton-catalyzed decomposition. However, uric acid is capable of scavenging hydroxyl radical (k = 7.2 × 109 M−1 s−1 [75]) and nitrogen dioxide (k = 1.9 × 107 M−1 s−1) [52] and, at the concentrations used in our assays, this scavenger

Fig. 4. Effect of uric acid and glutathione on the activity and nitration of SOD. (A and C) Activity of CuZnSOD (black bars) and MnSOD (white bars) (5 μM) after exposure to JU NO and JO2U− (10 μM/min, 10 min) in the absence and presence of uric acid (100 μM and 1 mM) or glutathione (1 and 10 mM) (1 mM uric acid and 1 mM GSH were only tested in MnSOD). (B and D) Immunodetection of 3-nitrotyrosine in samples of MnSOD exposed to J U NO and JO2U− in the absence and presence of uric acid (1 mM) or glutathione (10 mM) under the same conditions as in A and C. Asterisk denotes statistical difference respect to the control condition, using LSD statistical test (P < 0.05).

dependent manner (Fig. 1). However, we were not able to observe total inactivation, as was observed with larger concentrations of authentic peroxynitrite [47,48,50,51], in part because of the limitations of our system (i.e., changes in fluxes, accumulation of products, an enzyme that can interfere with the

Fig. 5. Effect of the variation of nitric oxide/superoxide flux ratios in the activity of CuZnSOD and MnSOD. (A) CuZnSOD (5 μM) was exposed to a constant JO2U− of 10 μM/min for 10 min (■) or 30 μM/min for 3 min (□), varying the JUNO rate of generation from 5 to 30 or 15 to 90 μM/min, respectively. Inset: Computer-assisted simulations of the steady-state levels of peroxynitrite at different JUNO/JO2U− ratios calculated using the reactions presented in Table 2. (B) MnSOD (5 μM) was exposed to a constant JO2U− (10 μM/min) for 10 min, varying the JUNO rate of generation from 5 to 30 μM/min. Inset: Immunodetection of protein- 3-nitrotyrosine in the samples.

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competes efficiently with tyrosine present in the enzyme. Thus, the lack of protection of enzyme inactivation by uric acid (Fig. 4A) clearly denotes that neither hydroxyl radical nor nitrogen dioxide are responsible of this process. As reported, MnSOD inactivation is related to specific tyrosine-34 nitration [47,48] and, in fact, in the samples treated with uric acid 3-nitrotyrosine is still observed (Fig. 4B). Due to the sustained inactivation, it can be assumed that the remnant nitration corresponds to nitrated tyrosine-34 and the protection exerted by uric acid is merely the inhibition of nitration of other tyrosines (Y-45 and Y-193), noncritical for the activity, by scavenging of nitrogen dioxide and hydroxyl radical. Moreover, and in support of this idea, MnSOD nitration by the horseradish peroxidase/nitrite/ hydrogen peroxide system leads to high levels of nitration with much less inactivation compared to peroxynitrite, in support again, of the nitration of noncritical tyrosines by nitrogen dioxide (not shown). Glutathione, which reacts directly with peroxynitrite as well as with peroxynitrite-derived radicals and fully protects SODs from authentic peroxynitrite, was not capable of fully protecting MnSOD from inactivation and nitration in the presence of nitric oxide and superoxide fluxes (Figs. 4C and D). This is due to the fact that at the concentrations of glutathione used in our assays (1 and 10 mM), a fraction of peroxynitrite is capable of reacting with the protein metal center to form an O = Mn4+, followed by the one-electron oxidation of adjacent tyrosine-34 to the corresponding tyrosyl radical.2 While in the case of authentic peroxynitrite the evolution of tyrosyl radical to 3-nitrotyrosine is efficiently inhibited by glutathione scavenging of nitrogen dioxide, in the presence of fluxes the persistence of tyrosine nitration suggests the involvement of a nitrogen dioxideindependent mechanism. In this regard, nitric oxide reaction with tyrosyl radical, leading to a transient 3-nitrosotyrosine intermediate which becomes further oxidized in two oneelectron steps by oxidized transition metals centers [72] (i.e., in our case O = Mn+4 formed from new molecules of peroxynitrite reacting with MnSOD), appears to be the most likely mechanism. In further support, while inactivation of MnSOD reached a plateau at nitric oxide/superoxide ratios over 2, in accordance with the calculated steady-state level of peroxynitrite (Fig. 5A, inset), tyrosine nitration continued to increase as levels of nitric oxide increased (Fig. 5B, inset). In the case of CuZnSOD, a protein radical was detected by immuno-spin trapping when exposing the enzyme to the fluxes. On a previous work [50] we detected the formation of a histidinyl radical, mediated by the reaction of the copper center with peroxynitrite, similar to that described with hydrogen peroxide [77,78]. Based on that result, we can infer the formation of histidinyl radical in our system, an amino acid-derived radical susceptible to immuno-spin trapping as recently reported [70]. In MnSOD, protein radical formation and tyrosine nitration were detected. MnSOD tyrosine nitration is one of the principal biomarkers of 2 Even though GSH can participate in tyrosyl radical repair [76], this would not be relevant for MnSOD tyrosine-34 as GSH does not have access to the narrow active site.

peroxynitrite formation in vivo and the observation of this modification by fluxes is an important event [41,47]. The radical observed by immuno-spin trapping in MnSOD can be attributed to tyrosyl radical, since the addition of DMPO to the reaction mixture diminished tyrosine nitration. MnSOD dimers were not observed after the exposure of the enzyme to the fluxes, in opposition to what has been previously reported with authentic peroxynitrite where in addition to 3-nitrotyrosine, 3-3′-dityrosine and higher molecular mass species of MnSOD were detected [48]. In conclusion, the more biologically relevant approach to SOD inactivation by peroxynitrite presented herein can help to understand some of the events that take place in the presence of elevated simultaneous fluxes of superoxide and nitric oxide. Specifically, this work provides a kinetic and mechanistic foundation for both the detection of nitrated and inactivated SOD and the overwhelming evidence about peroxynitrite formation in vivo despite the presence of this enzyme in tissues exposed to conditions such as sepsis, ischemia and reperfusion, and inflammation processes [39,42]. Moreover, with the data presented herein and including the modeling of the rapid intercellular diffusion of nitric oxide [36], it can be envisioned and predicted (Demicheli and Radi, preliminary data) how variations in SOD levels will modulate the signaling actions of a given flux of nitric oxide from a source to a target cell when superoxide is simultaneously produced as well as the amount of peroxynitrite formed and the subsequent oxidative processes. Note added in proof A report has just indicated that aerobic incubation of E. coli MnSOD with relatively high concentrations of nitric oxide can yield nitroxyl anion and secondarily peroxynitrite that can cause nitration and inactivation of the enzyme (Filipovic M.R., Stanic D., Raicevic S., Spasic M. and Niketic V. Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide. Free Radical Research. 42: 62-72 (2007)). Acknowledgments We thank Dr. Mónica Marín (Universidad de la República) for kindly providing an anti-human CuZn SOD antibody. V.D. is partially supported by a Ph.D. fellowship from PEDECIBA (Programa de Desarrollo de Ciencias Básicas, Uruguay). This work was supported by grants from the Howard Hughes Medical Institute to R.R., Comisión Sectorial de Investigación Científica, Universidad de la República to B.A., and Fondo Clemente Estable to C.Q. R.R. is a Howard Hughes International Research Scholar. References [1] McCord, J. M.; Fridovich, I. The reduction of cytochrome c by milk xanthine oxidase. J. Biol. Chem. 243:5753–5760; 1968. [2] Knowles, P. F.; Gibson, J. F.; Pick, F. M.; Bray, R. C. Electron-spinresonance evidence for enzymic reduction of oxygen to a free radical, the superoxide ion. Biochem. J. 111:53–58; 1969.

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