Homolytic Pathways Drive Peroxynitrite-Dependent Trolox C Oxidation

Chem. Res. Toxicol. 2004, 17, 1377-1384

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Homolytic Pathways Drive Peroxynitrite-Dependent Trolox C Oxidation Horacio Botti,† Madia Trujillo,† Carlos Batthyańy,† Homero Rubbo,† Gerardo Ferrer-Sueta,‡ and Rafael Radi*,† Departamento de Bioquı´mica and Center for Free Radical and Biomedical Research, Facultad de Medicina, and Laboratorio de Fisicoquı´mica Biolo´ gica, Instituto de Quı´mica Biolo´ gica, Facultad de Ciencias, Universidad de la Repu´ blica, Montevideo, Uruguay Received December 29, 2003

Peroxynitrite is a powerful oxidant implicated as a mediator in nitric oxide (•NO)- and superoxide (O2•-)-dependent toxicity. Peroxynitrite homolyzes after (i) protonation, yielding hydroxyl (•OH) and nitrogen dioxide (•NO2) free radicals, and (ii) reaction with carbon dioxide (CO2), yielding carbonate radical anion (CO3•-) and •NO2. Additionally, peroxynitrite reacts directly with several biomolecules. It is currently accepted that R-tocopherol is one important antioxidant in lipid compartments and its reactions with peroxynitrite or peroxynitrite-derived radicals may be relevant in vivo. Previous reports on the peroxynitrite reaction with Trolox C (TxOH)san R-tocopherol water soluble analoguessuggested a direct and fast reaction. This was unexpected to us as judged from the known reactivities of peroxynitrite with other phenolic compounds; thus, we thoroughly investigated the kinetics and mechanism of the reaction of peroxynitrite with TxOH and its modulation by CO2. Direct electron paramagnetic resonance studies revealed that Trolox C phenoxyl radical (TxO•) was the only paramagnetic species detected either in the absence or in the presence of CO2. Stopped-flow spectrophotometry experiments revealed a sequential reaction mechanism, with the intermediacy of TxO• and the production of Trolox C quinone (TxQ). Reactions were zero-order with respect to TxOH and first-order in peroxynitrite and CO2, demonstrating that the reaction of peroxynitrite with TxOH is indirect. In agreement, TxOH was unable to inhibit the direct peroxynitrite-mediated oxidation of methionine to methionine sulfoxide. TxOH oxidation yields to TxO• and TxQ with respect to peroxynitrite were ∼60 and ∼31%, respectively, and increased to ∼73 and ∼40%, respectively, in the presence of CO2. At peroxynitrite excess over TxOH, the kinetics and mechanism of oxidation are more complex and involve the reactions of CO3•- with TxO• and the possible intermediacy of unstable NO2-TxOH adducts. Taken together, our results strongly support that H+- or CO2-catalyzed homolysis of peroxynitrite is required to cause TxOH, and hence, R-tocopherol oxidation.

Introduction Peroxynitrite1 is a strong oxidizing and nitrating agent that can be generated in vivo by the reaction of superoxide (O2•-) and nitric oxide (•NO) radicals (k ) 4.3-16 × 109 M-1 s-1) (1-6), outcompeting the controlling steps for •NO and O2•- metabolism and/or detoxification (7). Peroxynitrite-mediated oxidations may follow peroxynitrite protonation and subsequent peroxo-bond homolysis (k ) 4.5 s-1, 37 °C) to yield hydroxyl (•OH) and nitrogen dioxide (•NO2) radicals in a ∼30% yield (range, 24-32%) (8-18). Nevertheless, the majority of peroxynitrite participates in vivo in direct bimolecular processes, thus minimizing •OH-mediated oxidations (19, 20). Importantly, peroxynitrite anion directly reacts with CO2 * To whom correspondence should be addressed. Tel: (5982)9249561. Fax: (5982)924-9563. E-mail: [email protected]. † Departamento de Bioquı´mica and Center for Free Radical and Biomedical Research. ‡ Laboratorio de Fisicoquı´mica Biolo ´ gica. 1 IUPAC recommended names for peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH) are oxoperoxonitrate (1-) and hydrogen oxoperoxonitrate, respectively. Unless otherwise stated, the term peroxynitrite is used in this work to refer to the sum of ONOO- and ONOOH.

[k ) 5.8 × 104 M-1 s-1, 37 °C (21)] yielding a shortlived species, presumably nitrosoperoxocarboxylate (ONOOCO2-, 1-carboxylato-2-nitroso-dioxidane) (22-25), which homolyzes to the oxidizing radicals carbonate anion (CO3•-) (22-25) and •NO2 with a reported yield of ∼35% (range, 19-40%) (16, 25-28). R-Tocopherol (R-TOH)2 may be the most important reductant of lipid compartments. R-TOH and Rtocopheroxyl radical break radical chain reactions in biomembranes and lipoproteins by electron transfer and addition reactions with lipid peroxyl radicals, respectively (29-31). In addition, R-TOH and γ-tocopherol (γ-TOH) have attracted attention as potential antioxidants against peroxynitrite and •NO2 in lipid milieu (32-37). It has been previously demonstrated that R-tocopheryl quinone and Trolox C quinone (TxQ) are the major stable products formed from peroxynitrite-mediated oxidation of R-TOH and Trolox C (TxOH, a water soluble R-TOH analogue) (34) and also that peroxynitrite and •NO2 can induce 2 Abbreviations: R-TOH, R-tocopherol; γ-TOH, γ-tocopherol; TxOH, Trolox C; TxO•, Trolox C radical; TxQ, Trolox C quinone; TxKD, Trolox C ketodienone; dtpa, diethylenetriaminepentaacetic acid; metSdO, methionine sulfoxide; DMSO, dimethyl sulfoxide; PITC, phenylisothiocyanate.

10.1021/tx034269i CCC: $27.50 © 2004 American Chemical Society Published on Web 09/28/2004

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oxidation and nitration of γ-TOH-generating 5-nitro-γTOH (36, 37). Previous reports have not unambiguously addressed the relative contribution of direct vs radicalmediated oxidation pathways in peroxynitrite-mediated chromanol oxidation, particularly in the oxidation of the fully substituted chromanols such as R-TOH and TxOH (34, 38). In fact, it has been reported that peroxynitrite directly reacts with TxOH resulting in competing fast one- (k ) 1.8 × 105 M-1 s-1, 25 °C, pH 7) and two-electron (k ∼ 600 M-1 s-1, 25 °C, pH 7) oxidations (38). This was unexpected to us as judged from the known reactivities of peroxynitrite with phenolic compounds (39-43), and we suspected that confounding experimental factors arising when studying peroxynitrite reaction kinetics could be responsible for the rather large rate constants reported. In addition, it has been assumed that peroxynitrite constitutes a two-electron nonradical oxidant for R-TOH in low-density lipoprotein (LDL) (44, 45). In contrast, we have recently reported that peroxynitriteinduced R-TOH oxidation in LDL proceeds through a oneelectron free radical pathway strongly modulated by nitric oxide, ascorbate, and carbon dioxide (46). In this work, we investigated the reaction kinetics and mechanism of the reaction of peroxynitrite with TxOH as well as its modulation by CO2. We hypothesize that TxOH and, hence, R-TOH oxidation occur exclusively via homolysis of the peroxo-bond in ONOOH and ONOOCO2to free radical species.

Experimental Procedures Materials. TxOH (6-hydroxy-2,5,7,8-tetramethylchroman-2caboxylic acid) (97%) was from Aldrich Chem Co. (Milwaukee). The rest of the chemicals were of analytical grade and were used as received from Sigma-Aldrich Co. (St. Louis, MO). All solutions were prepared with deionized water using a water deionization system from Barnstead/Thermolyne (Dubuque, IA). TxOH stock solutions (10-50 mM) were prepared just before use by dissolving in alkaline (pH ∼9) phosphate (50-150 mM), adjusting the final pH to ∼7.3. To prevent metal-catalyzed oxidations, 100 µM diethylenetriaminepentaacetic acid (dtpa) was included in some experiments. Peroxynitrite was synthesized, handled, and quantitated as previously described (19, 47). Contaminating nitrite in freshly prepared stock solutions was less than 30% of peroxynitrite, as determined using the Griess reaction. Residual H2O2 was minimized by incubation with manganese dioxide for 6 h at 4 °C before use. To prevent carbonate contamination, only freshly prepared sodium or potassium hydroxide stock solutions were used (48). Electron Paramagnetic Resonance (EPR) Measurements. Continuous flow EPR spectroscopy studies were carried out using a X band spectrometer equipped with a standard ER 4119HS cavity (Bruker BioSpin Corporation). Detection settings used in this work were similar to those reported previously for Trolox C radical (TxO•) (49); specifically: gain, 1 × 105; modulation amplitude, 0.5 G; time constant, 163.8; field center, 3483 G; scan range, 80 G; microwave power, 19.97 mW; and microwave frequency, 9.76 GHz. The continuous flow setup consisted of two syringes adjusted to a syringe pump model 355 (SAGE Instruments) connected to a 20 µL mixing chamber and finally to the entrance of a flat quartz cell. The dead volume was ∼500 µL, and flow rates ranged from 0.5 to 50 mL min-1. All EPR experiments were performed at room temperature, in aerobic conditions, and at pH 7.40. Stopped-Flow Determinations. Most stopped-flow measurements were carried out using an Applied Photophysics SF.17MV spectrophotometer (Leatherhead, England) with a mixing time of ∼1 ms and a 1 cm optical path. Some experiments were performed in a UV-vis Cary 50 spectrophotometer (Vari-

Botti et al. an) using a RX.2000 rapid kinetics accessory from Applied Photophysics. This setup allowed accurate detection at the shorter wavelengths employed (240 and 250 nm). Equal volumes of peroxynitrite in ∼10 mM NaOH and TxOH (0-20 mM) in 100 mM potassium phosphate buffer, initial pH 7.20, were prepared by dilution of stock solutions directly into the syringe and immediately mixed without any noticeable change of initial peroxynitrite concentration between runs. Stock bicarbonate solutions (25 mM) in potassium phosphate buffer, pH ∼7.2, was prepared and used within an hour. When bicarbonate was included, ∼2 min was allowed for equilibration in the syringe at 37 °C before mixing. The peroxynitrite disappearance was followed at 302 nm as previously described (19). The peroxynitrite disappearance in the presence of TxOH could not be directly assessed because of difficulties in interpretation of complex absorbance changes in the 250-330 nm region, where peroxynitrite- and TxOH-derived reaction intermediates absorb. Instead, an initial rate approach (50) was used following the formation of TxO• [438 nm ) 6030 M-1 cm-1 (49, 51)]. In addition, TxQ was determined spectrophotometrically [266 nm ) 19000 M-1 cm-1 (34)]. Data corresponding to 4-20 ms were used to determine the initial rate of TxO• formation (V0). All stoppedflow experiments were performed at 37 ( 0.5 °C. The final pH was always measured at the outlet (pH 7.40 ( 0.05). TxQ Yield. It has been previously reported that the TxQ is the major end product of peroxynitrite-induced TxOH oxidation (34). The yields of TxQ formation after a 5 min TxOH incubation with peroxynitrite were determined either by reverse phase HPLC with UV-vis detection as previously described (34) or by UV-vis absorption spectra in a Shimadzu UVPC 2410 spectrophotometer according to the following equation:

[TxQ] ) [Abs266nm - (Abs310nm TxOH266nm/TxOH310nm)]/TxQ266nm (1) Peroxynitrite was added as a bolus using a 25 µL gastight Hamilton syringe with vigorous vortex mixing during additions. Incubations were performed at room temperature in 100 mM phosphate buffer with 100 µM dtpa at pH 7.40 ( 0.05. Competition Kinetics and Methionine Sulfoxide (MetSdO) Determinations. Methionine was selected for competition kinetic experiments because the kinetics and mechanism of reaction with peroxynitrite have been already studied (40, 42, 52). Peroxynitrite directly reacts with methionine (k ) 180 ( 8 M-1 s-1, 25 °C, pH 7.4) (42). The reaction results in methionine oxidation to MetSdO with the concomitant formation of nitrite with near 66% yield with respect to peroxynitrite as well as peroxynitrite isomerization to nitrate without methionine oxidation in approximately 33% yield (52). It is debated if peroxynitrite-derived radicals can also react with methionine to yield ethylene (42, 52). Nevertheless, this radical pathway can be minimized by selecting the appropriate starting target concentrations (50). At 15 mM methionine, free radicalmediated oxidations were reduced to less than 10% with respect to the initial concentration of peroxynitrite (e400 µM). In addition, methionine oxidation to the sulfoxide form can be catalyzed by metal traces (53). This was minimized by including 100 µM dtpa in reaction buffers as well as in methionine and TxOH stock solutions. MetSdO was determined by gradient reverse phase HPLC with UV detection after derivatization with phenyl-isothiocyanate (PITC) (54). MetSdO identification was based on coelution with a standard synthesized by incubation of methionine with excess dimethyl sulfoxide (DMSO) in acidic media, as previously reported (55). Nitric Oxide Measurement. Nitric oxide formation from TxOH and peroxynitrite in aerobic conditions was quantitated by electrochemical detection using a •NO sensor (Iso-NO, WPI Inc., Sarasota, FL). Electrode response calibration was done by measuring •NO produced from NO2- reduction by KI/H2SO4. All measurements were performed at 37 °C and pH 7.4 ( 0.05.

Peroxynitrite-Dependent Trolox C Oxidation

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Results TxO• Formation during Peroxynitrite-Dependent TxOH Oxidation. TxOH oxidation was studied by continuous flow EPR (Figure 1A). The observed paramagnetic signal is consistent with the formation of the radical of TxOH (TxO•). The same EPR signal was obtained when TxOH was exposed to peroxynitrite in the presence of CO2, indicating that TxO• was also formed under this condition (Figure 1A). There was no EPR signal when either TxOH or peroxynitrite was omitted (not shown). The hyperfine splitting constants determined in this work (3H, 5.21 G; 3H, 3.80 G; 3H, 0.94 G; and 2H, 0.24 G) are in agreement with previous reports (49, 56, 57) and were utilized for spectrum simulation (Figure 1A, lower trace). The reaction of TxOH with peroxynitrite was also studied by stopped-flow spectrophotometry. Time courses of TxO• and TxQ transient concentrations are shown (Figure 1B). Either in the absence or in the presence of CO2, TxO• increases to a maximum and then decays, while the TxQ concentration time course exhibits an initial lag phase before accumulation. In fact, the maximal TxO• concentration immediately precedes the maximal rate of TxQ formation. Notably, CO2 accelerated and enhanced both TxO• and TxQ formation (Figure 1B). On the other hand, DMSO halved TxQ yield if used at high enough concentrations (∼100 mM) to scavenge all peroxynitrite-derived •OH (Figure 1C). Determination of the Reaction Order in TxOH and Peroxynitrite. Stopped-flow experiments were performed to determine the order of reaction with respect to [TxOH]. The initial peroxynitrite concentration, pH, and temperature remained constant while the initial TxOH concentration was varied (0-10 mM). Higher transient [TxO•] were observed with increasing [TxOH]0 (Figure 2A) with maximal [TxO•] approaching saturation at the highest tested [TxO]0 (Figure 2A, inset). Accordingly, the initial rates of TxO• formation determined between 4 and 20 ms (V0) increased hyperbolically reaching a maximum limit of 89 µM s-1 at infinite [TxOH]0 concentration (Figure 2B), representing 59% of the rate of peroxynitrite disappearance determined at 302 nm in the absence of TxOH at the same [ONOO-]0. These results demonstrate that the initial rate of formation of TxO• is zero-order with respect to [TxOH]0 during peroxynitrite-induced TxOH oxidation and that the predominant oxidative pathway is a one-electron free radical oxidation to yield TxO• provided that TxOH is in large excess (∼100 times) over peroxynitrite. The yield of TxQ formation as a function of the [TxOH]0 was studied by spectrophotometric and RP-HPLC analysis (Figures 2C and 4C). TxQ formation increased linearly at low [TxOH]0 but plateaus with a yield of ∼23% with respect to [ONOO-]0 at [TxOH]0 ) 1 mM. At low [TxOH]0, approximately 82% of TxOH had been oxidized to TxQ and ∼18% remained as the reduced form (TxOH), although peroxynitrite was in large excess relative to [TxOH]0 (Figure 2C). At [TxOH]0 ) 10 mM, the yield of TxQ increased to ∼30% (Figure 4C and Table 1). To determine the order of the reaction with respect to peroxynitrite, we analyzed TxO• formation as a function of [ONOO-]0 by stopped-flow spectrophotometry, with constant [TxOH]0 and variable [ONOO-]0 (Figure 3). It was observed that V0 had a linear dependence on [ONOO-]0 at all [TxOH]0 tested, evidencing that the rate

Figure 1. TxO• and TxQ formation during peroxynitriteinduced TxOH oxidation. (A) EPR spectra. A continuous flow technique was employed; solutions containing 1 mM peroxynitrite in 2 mM NaOH and 5 mM TxOH in 50 mM phosphate, pH 7.30, at room temperature, either in the absence (upper trace) or in the presence (middle trace) of CO2 (300 µM), were pumped to the detection cell after mixing. The flow was 2.4 mL min-1, and the final pH was 7.40 ( 0.03. Simulated spectrum (lower trace) fitting to the hyperfine splitting constants obtained from experimental data using WinSim V1 downloaded from the National Institute of Environmental Health Sciences Public EPR Software Tools (http://EPR.niehs.nih.gov/pest.html). (B) Stopped-flow time courses of TxO• (λ ) 438 nm, traces a and c) and TxQ (λ ) 266 nm, traces b and d) during peroxynitrite (175 µM)-induced TxOH (5 mM) oxidation in the absence (traces a and b) and presence (traces c and d) of CO2 (100 µM). (C) Stopped-flow time courses of TxQ accumulation after peroxynitrite (400 µM)-induced oxidation of TxOH (300 µM) in the absence (solid line) and presence (dash line) of 0.1 M DMSO. Experimental conditions: T ) 37 ( 0.1 °C, pH 7.40 ( 0.05.

of TxO• formation is first-order in peroxynitrite concentration. Again, at the higher [TxOH]0 tested, the slope of the curve (kapp) approached 60% of the first-order rate constant of peroxynitrite disappearance at 37 °C and pH 7.4 (Figure 3, dotted line). Effect of TxOH on Direct Target Oxidation by Peroxynitrite. Competition experiments were performed to discard any possible direct bimolecular reaction between peroxynitrite and TxOH. Methionine was selected as the alternative target for peroxynitrite. The yields of MetSdO based on [ONOO-]0 (0-200 µM) with methionine in excess (15 mM) were 58 ( 1 and 59 ( 2% in the absence and presence of TxOH (40 mM), respectively (not shown).

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Figure 3. TxO• formation as a function of initial peroxynitrite concentration. Plot of initial rates of TxO• formation between 4 and 20 ms as a function of [ONOO-]0 at different [TxOH]0 [0.3 (9) and 5.0 (2) mM]. Dotted line: Initial rate of formation of peroxynitrite-derived radicals calculated from the initial rates of peroxynitrite decay and the reported yield of radicals. Mean ( SD, n g 7. Experimental conditions: T ) 37 ( 0.1 °C, pH 7.40 ( 0.05.

Figure 2. TxOH oxidation as a function of initial TxOH concentration. (A) Stopped-flow spectrophotometric determination of TxO• transient concentration during the first 20 s of peroxynitrite (160 µM)-induced TxOH (0.3, 0.5, 1.0, 2.0, 5.0, and 10 mM; traces a-f, respectively) oxidation. Each trace is the averaged data of at least seven repetitions of a representative experiment: T ) 37 ( 0.1 °C, pH 7.40 ( 0.05. Inset: Maximal TxO• transient concentration as a function of [TxOH]0; hyperbolic function fit to data. (B) The rates of TxO• formation between 4 ms e t e 20 ms obtained from the same experiment as in panel A plotted as a function of TxOH initial concentrations. Solid line: hyperbolic function fit to data. (C) Spectrophotometric determination of the TxQ final concentration after peroxynitrite (200 µM) reaction with different initial concentrations of TxOH (0-1.0 mM). Data represent means ( SD (n ) 3): T ) 25 °C, pH 7.40 ( 0.03. Table 1. TxO• and TxQ Yields with Respect to Peroxynitritea condition ONOO-

ONOO- + CO2

TxO•b (%)

TxQc (%)

60 ( 2 73 ( 1

31 ( 1 40 ( 1

a [TxOH] g 5 mM; 150 µM e [ONOO-] e 200 µM; phosphate 0 0 buffer (50-100 mM); T ) 37 °C, pH 7.4. b Stopped-flow determinac tions. End point HPLC determinations.

Carbon Dioxide Modulation of PeroxynitriteInduced TxOH Oxidation. TxO• and TxQ formations from TxOH and peroxynitrite in the presence of CO2 were studied with more detail (Figure 4). Stopped-flow experiments showed that the peak [TxO•] transient increases and reaches saturation with increasing [CO2]0 (Figure 4A and inset) and that this was accompanied by increased rates of TxO• formation and decay (Figure 4B,A, respec-

Figure 4. Carbon dioxide modulation of peroxynitrite-dependent TxOH oxidation. (A) TxO• time courses during peroxynitrite (200 µM)-induced TxOH (5 mM) oxidation in the presence of different CO2 concentrations (0, 25, 50, 75, 100, 150, 200, and 250 µM). Inset: Maximal transient TxO• concentrations as a function of [CO2]0. (B) Initial rates of TxO• formation plotted against CO2 concentrations. (C) TxQ formation following 1 (9) and 10 mM (b) TxOH reaction with peroxynitrite (200 µM) in the presence of different concentrations of CO2. Experimental conditions: T ) 37 °C, pH 7.40 ( 0.05. Means ( SD are displayed.

tively). The initial rate of TxO• formation linearly depended on [CO2]0 at the lowest [CO2]0. A slope of ∼6.6 s-1 was derived from this plot (Figure 4B), allowing determination of the yield of TxO• at [TxOH]0 ) 5 mM


Figure 5. TxOH oxidation at high peroxynitrite to TxOH ratios. (A) Time courses of TxO• formed with increasing peroxynitrite concentrations (a, 50; b, 100; c, 200; and d, 400 µM. Inset: e, 500; and f, 1000 µM) and constant [TxOH]0 (300 µM). (B) Absorbance changes at the indicated wavelengths after mixing 300 µM TxOH with 400 µM peroxynitrite. (C) TxO• traces at 438 nm during the reaction of TxOH (300 µM) with peroxynitrite (400 µM) in the absence (trace a) and presence of 2 mM CO2 (trace b) or 100 mM DMSO (trace c). Arrows denote two regeneration phases of TxO•. Representative results of five experiments are shown.

(Table 1). The initial rate of TxO• formation was a fraction of the initial rate of peroxynitrite decay as a function of [CO2]0, which showed a linear dependence on CO2 in the whole range of [CO2]0 used (Figure 4B, dotted line). Accordingly, the yield of TxQ also increased in the presence of CO2 (Table 1), reaching saturation at [ONOO-]0 ∼ [CO2]0 at either 1 or 20 mM [TxOH]0 (Figure 4C). TxOH Oxidation at High [ONOO-]0 to [TxOH]0 Ratios. Stopped-flow spectrophotometry (Figure 2A) and continuous flow EPR experiments (not shown) supported that TxO• decays with second-order kinetics if TxOH is in excess over peroxynitrite, in agreement with the already established dismutation reactions of TxO• (51). Nevertheless, if TxOH is not in large excess, the maximal rate of TxO• disappearance increases almost linearly with [ONOO-]0 (Figure 5A and Supporting Information) but the maximal transient [TxO•] remains almost unchanged at [ONOO-]0 g 200 µM (Figure 5A, traces c-f and inset). Thus, under these conditions, TxO• decay cannot be fully explained by its disproportionation reactions only, implying that other pathways begin to participate. Moreover, a second phase of TxO• formation (which we will refer as

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TxO• “regeneration”) was evident if peroxynitrite was in excess over TxOH (Figure 5A and inset). We compared absorbance changes at four wavelengths to follow TxO• (438 nm), TxQ (266 nm), and any intermediate with the UV absorption characteristics similar to those of Trolox C ketodienone (TxKD) and tocopherones (240 nm) (33, 51) and their isosbestic point with quinones (250 nm) (Figure 5B). We observed that the maximal rate of absorbance increase at 240 nm exactly coincided with the first maximum of [TxO•], supporting that TxO• was being converted to a species absorbing at this wavelength. Also, we observed that the maximal rate of absorbance decrease at 240 nm was simultaneous with the second relative maximum of TxO• (regenerated TxO•). In addition, absorbance changes at 250 nm supported that intermediates with absorbance at 240 nm were being quantitatively converted to quinones only after TxO• regeneration. Finally, we observed that the initial decay of TxO• was associated with a decrease of the rate of TxQ formation (Supporting Information). To evaluate the role of •OH, CO3•-, and •NO2, we modulated the reaction with DMSO, CO2, and CO2 plus NO2-. TxO• regeneration was evident in the absence (Figure 5C, trace a) as well as in the presence of 100 mM DMSO (Figure 5C, trace c). The time course of TxO• in the presence of 2 mM CO2 showed an early phase of TxO• regeneration (Figure 5C, trace b), which was not evident in the previously tested conditions. If NO2- (50 mM) was added along with 2 mM CO2, the maximum [TxO•] decreased but the extent and timing of the two regeneration phases remained unchanged (not shown). Finally, we found no •NO production from the reactions/decomposition of any intermediate (not shown). Altogether, the results shown in Figure 5 and computerassisted simulations (Supporting Information) indicate the formation of at least two kinetically distinguishable intermediates capable of regenerating TxO• from the reactions of TxO• with •NO2.

Discussion TxO• Is an Obligatory Intermediate of Peroxynitrite and Peroxynitrite Plus CO2-Dependent TxQ Formation. TxO• was shown to be formed in the absence and in the presence of CO2, suggesting that similar mechanisms are underlying. Stopped-flow experiments showed that peak TxO• concentration immediately precedes the maximal rate of TxQ formation irrespective of the absence or presence of CO2, evidencing that in both cases the mechanism of oxidation of TxOH to TxQ is sequential (one-electron at a time) and involves TxO• as an obligatory intermediate. This conclusion is different to early proposals suggesting that TxO• formations during peroxynitrite-induced TxOH and R-TOH oxidation are minor pathways in the way to quinone accumulation (34) and strongly contrasts with the view of peroxynitrite as a two-electron R-TOH oxidant (45). Peroxynitrite-Dependent TxOH Occurs Via Peroxo-Bond Homolysis in ONOOH and ONOOCO2-. We studied the dependence of TxO• formation rate on [TxOH]0 and demonstrated that the rate of the reaction is zero-order in [TxOH]0; hence, TxO• formation is not dependent on a bimolecular step involving peroxynitrite and TxOH. Additionally, we showed that under TxOH excess, TxQ is formed exclusively via TxO• dismutation and that the maximal yield of TxQ is half of that of TxO•

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and ∼30% of [ONOO-]0 (Table 1); therefore, TxQ formation does not depend on a direct reaction of TxOH with peroxynitrite. Finally, in agreement with a previous report (42), we observed that Trolox C does not compete with methionine for peroxynitrite, since it did not affect MetSdO formation. Therefore, in contrast with previous work (38), we conclude that a bimolecular process involving TxOH and peroxynitrite can be ruled out. The yields of TxO• and TxQ are in agreement with the higher reported values of free radical yield from ONOOH and ONOOCO2- homolysis (8-18, 25-28); therefore, •NO2, • OH, and CO3•- radicals are responsible for peroxynitritedependent TxOH oxidation. Similarly, R-TOH would not behave as a direct peroxynitrite scavenger in vivo, as further supported by our previous work on the mechanism of peroxynitrite-dependent R-TOH oxidation in LDL (46). TxO• Formation and Decay Pathways at High [ONOO-]0 to [TxOH]0 Ratios. Stopped-flow analysis show that TxO• dismutation is not the only pathway of TxO• oxidation to TxQ at low [TxOH]0/[ONOO-]0 ratios. Comparison of experimental results and computer-assisted simulations supported the parallel oxidation of TxO• by peroxynitrite-derived radicals and discarded a direct TxO• oxidation by peroxynitrite itself (Supporting Information). Also, in these conditions, the apparent initial rate of TxO• formation departs very early in the course of the reaction from an initial rate behavior (Figure 2B). Because this is not due to TxOH or peroxynitrite depletion, it implies rapid reaction of intermediates. A constant fraction of TxOH remained unoxidized at the end of the reaction although peroxynitrite was in large excess. In addition, we evidenced two TxO• regeneration processes. We tentatively ascribe these phenomena to the decomposition of at least two intermediates formed through a fast and at least partially reversible reaction of TxO• and •NO2 (Scheme 1 and Supporting Information). A detailed study of this reactivity including isolation and structural characterization of intermediates and determination of the rate constants involved was beyond the aims of this study but may be of relevance to better understand the role of totally (R-TO• and TxO•) and partially (i.e., γ-TO•) substituted chromanoxyl radicals in •NO and •NO2 metabolism and toxicity. However, plausible intermediates are nitrogen-containing adducts of •NO2 at the phenolic oxygen and 8a-position, the latter being possibly more stable than the former (33, 36). The instability of these proposed intermediates explains why TxQ is the major product and is in agreement with previous reports showing that unlike peroxynitritemediated oxidation of γ-TOH (58) and unsubstituted phenols (59, 60) the oxidation of TxOH and R-TOH results exclusively in quinone formation (34). Additionally, nitrite contamination present in all peroxynitrite preparations efficiently competes with TxOH for •OH at low [TxOH]. This might suggest that the low yields of TxOH oxidation at low [TxOH] may be due to the lack of participation in this process of •NO2, as previously proposed (38). In contrast, in this work, we show that even at the lowest tested [TxOH]0, high [DMSO]0, a well-characterized •OH scavenger (k ) 7 × 109 M-1 s-1) (61-63), exactly halved the yield of TxQ showing that both •OH and •NO2 can participate in TxOH oxidation. Finally, the •OH reaction with peroxynitrite anion [k ) 4.8 × 109 M-1 s-1 (64)] can also compete with TxOH oxidation.

Botti et al. Scheme 1. One-Electron Pathways of Peroxynitrite-Dependent TxOH Oxidation

Carbon Dioxide Modulation of PeroxynitriteDependent Chromanol Oxidation. The role of CO2 deserves a special consideration since it strongly modulates peroxynitrite biochemistry (20, 65-67). Previous studies support that CO2 limits the oxidizability of intraand transmembrane targets by peroxynitrite and peroxynitrite-derived radicals (65, 68-70). One explanation is that CO3•- is a charged species in vivo (pKa < 0) (71), having a low ability to penetrate and diffuse into hydrophobic compartments. Thus, although in this work we show that CO2 enhances and accelerates TxOH oxidation, we have recently reported that it decreases lipid and concomitantly R-TOH oxidation in LDLs exposed to peroxynitrite (46), underscoring the relevance of the diffusional properties of peroxynitrite and peroxynitritederived radicals when extrapolating reaction chemistries from homogeneous to compartmentalized systems. Biomedical Relevance. This work was performed with the understanding that key aspects of peroxynitrite reactivity toward TxOH can be extrapolated to the reactivity of peroxynitrite with R-TOH in membranes and lipoproteins. In this regard, it has been demonstrated that R-carboxyethyl-6-hydroxychroman (a natural metabolite of R-TOH lacking the phytyl side chain) has a similar reactivity toward oxidizing free radicals that parent vitamin E and TxOH (72, 73). Like vitamin E, TxOH has been successfully used as a synthetic antioxidant in some animal models of oxidative stress and may be of potential pharmacological usage (74-76). Moreover, an antioxidant role for water soluble metabolites of TOHs in vivo has been suggested, particularly during vitamin E supplementation (72).


The involvement of peroxynitrite in a wide range of pathophysiological processes, including cardiovascular and neurodegenerative diseases (77), has impelled the current investigation on natural (78-80) and synthetic compounds (81-84) as peroxynitrite scavengers. This possible pharmacological strategy is based in peroxynitrite interception and inactivation by means of its direct chemical reaction/s with the scavenger. From this perspective, knowledge of the kinetics and mechanism of peroxynitrite-dependent TxOH oxidation provides clues to understand the protective role of natural as well as synthetic chromanol compounds against peroxynitritedependent oxidative damage. This work supports that TxOH, and by extension R-TOH, can inhibit peroxynitrite-derived radical-induced oxidations. In this process, there is no direct scavenging of peroxynitrite and TxO• is an obligatory intermediate.

Acknowledgment. H.B. and C.B. were partially supported by fellowships from the Programa para el Desarollo de las Ciencias Ba´sicas and Comisioń Sectorial de Investigacioń Cientı´fica, Universidad de la Repu´blica, Uruguay. This work was supported by grants from the Howard Hughes Medical Institute and the Guggenheim Foundation to R.R. and H.R. and from Fundacioń Manuel Pe´rez to C.B. The contribution of the National Institutes of HealthsFogarty and Wellcome Trust grants is also gratefully acknowledged. R.R. is an International Research Scholar of the Howard Hughes Medical Institute. We thank S. Witkowski for helpful comments. Supporting Information Available: Computer-assisted simulations of transient TxO• concentrations under conditions similar to those of the experiments shown in Figure 5 assuming different kinetic and mechanistic models and a table of reactions and rate constants. This material is available free of charge via the Internet at http://pubs.acs.org.

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Homolytic Pathways Drive Peroxynitrite-Dependent Trolox C Oxidation

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