The Mechanism of the Peroxynitrite-Mediated Oxidation of Myoglobin

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The Mechanism of the Peroxynitrite-Mediated Oxidation of Myoglobin in the Absence and Presence of Carbon Dioxide Susanna Herold,* Michael Exner, and Francesca Boccini Laboratorium fu¨ r Anorganische Chemie, Eidgeno¨ ssische Technische Hochschule, ETH Ho¨ nggerberg, CH-8093 Zu¨ rich, Switzerland Received August 7, 2002

Among the cellular components that can react directly with peroxynitrite in the presence of physiological CO2 concentrations are sulfur-, selenium-, and metal-containing proteins, in particular hemoproteins. We have previously shown that the reactions of peroxynitrite with oxymyoglobin (MbFeO2) and oxyhemoglobin proceed via the corresponding ferryl species, which, in a second step, are reduced to the iron(III) forms of the proteins (metMb and metHb). In this study, we have investigated the influence of the concentration of added CO2 on the kinetics and the mechanism of the peroxynitrite-mediated oxidation of MbFeO2. We found that this reaction proceeds in two steps via the formation of MbFeIVdO also in the presence of millimolar amounts of CO2. As compared to the values measured in the absence of added CO2, the secondorder rate constant for the first reaction step in the presence of 1.2 mM CO2 is significantly larger [(4.1 ( 0.7) × 105 M-1 s-1, at pH 7.5 and 20 °C], whereas that for the peroxynitritemediated reduction of MbFeIVdO to metMb is almost unchanged [(3.2 ( 0.2) × 104 M-1 s-1, at pH 7.5 and 20 °C]. Finally, we show that because of the parallel decay of peroxynitrite, 8-25 equiv are needed to completely oxidize MbFeO2 to metMb, with larger amounts required to oxidize diluted MbFeO2 solutions in the presence of CO2. Simulation of the reaction in the absence and presence of CO2 was carried out to get a better understanding of the mechanism. The results suggest that CO3•- and NO2• may be involved in the reaction and interact with MbFeO2 and MbFeIVdO, respectively.

Introduction Nitrogen monoxide and superoxide react at a nearly diffusion-controlled rate to form peroxynitrite1 (1). This strong oxidizing and nitrating agent has been suggested to be formed in biological environments where NO• and O2•- are simultaneously generated in large concentrations (2). Superoxide can be produced through enzymes such as NADPH2 oxidase and xanthine oxidase or via electron leakage in the mitochondrial respiration chain (3). Large concentrations of NO• (up to µM) can be generated from iNOS, for instance, in inflammatory sites (4). The reaction between peroxynitrite and CO2, commonly present in tissues in millimolar concentrations, represents a key route of peroxynitrite consumption in biological systems. One of the effects of CO2 is to strongly reduce the lifetime of peroxynitrite, thus partially preventing membrane crossing and limiting its radius of action. The reaction between the peroxynitrite anion and CO2 is quite fast [6 × 104 M-1 s-1 at 37 °C (5) and 3 × 104 M-1 s-1 at 24 °C (6)] and yields the adduct 1-carboxylato-2-nitroso* To whom correspondence should be addressed. Fax: 41(1)632 10 90. E-mail: [email protected]. 1 The recommended IUPAC nomenclature for peroxynitrite is oxoperoxonitrate(1-); for peroxynitrous acid, it is hydrogen oxoperoxonitrate. The term peroxynitrite is used in the text to refer generically to both oxoperoxonitrate(1-) (ONOO-) and its conjugate acid, hydrogen oxoperoxonitrate (ONOOH). 2 Abbreviations: Hb, hemoglobin; HbFeO , oxyhemoglobin; Mb, 2 myoglobin; MbFeO2, oxymyoglobin; metMb, iron(III)myoglobin; MbIV Fe dO, oxoiron(IV) myoglobin, ferryl myoglobin; MbFeII, deoxymyoglobin; NADPH, nicotinamide adenine dinucleotide phosphate; iNOS, inducible nitric oxide synthase.

dioxidane ONOOCO2- (5, 7). As compared to peroxynitrite, ONOOCO2- is a stronger nitrating agent but less reactive toward thiols (6, 8, 9). In the absence of substrates, ONOOCO2- rapidly decays to nitrate and CO2. This reaction has been proposed to partly proceed by homolysis of the peroxo bond and thus generate nitrogen dioxide and the carbonate radical [CO3•-; systematic name, trioxidocarbonate(•1-)]. The latter radical has been detected by several groups, but its quantification is still a matter of disagreement (7, 10-14). Among the cellular components that can react directly with peroxynitrite in the presence of physiological CO2 concentrations are sulfur- (15-17), selenium- (18-20), and metal-containing proteins (18-20), in particular hemoproteins. We have recently investigated the reaction of peroxynitrite with MbFeO2 and HbFeO2 (21). We found that the reaction rates are comparable to that of the reaction between peroxynitrite and CO2. The secondorder rate constants for the peroxynitrite-mediated oxidation of MbFeO2 and HbFeO2 are (5.4 ( 0.2) × 104 M-1 s-1 (at pH 7.3 and 20 °C) and (8.8 ( 0.4) × 104 M-1 s-1 (at pH 7.0 and 20 °C), respectively (21). For both proteins, reaction with peroxynitrite first leads to the formation of the corresponding ferryl species. In a second step, MbFeIVdO and HbFeIVdO are reduced to the corresponding iron(III) forms of the proteins (metMb and metHb). The rate constants for these processes are (2.2 ( 0.1) × 104 M-1 s-1 (at pH 7.3 and 20 °C) and (9.4 ( 0.7) × 104 M-1 s-1 (at pH 7.3 and 20 °C), respectively (21). In addition, we have determined the second-order rate constant for the two electron oxidation of deoxyMb by

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Mechanism of the Oxidation of Myoglobin by Peroxynitrite

peroxynitrite: k ≈ 106 M-1 s-1 (at pH 7.5 and 20 °C) (21). This reaction is probably one of the fastest bimolecular reactions peroxynitrite can undergo with biomolecules. Here, we report further mechanistic investigations of the reaction between MbFeO2 and peroxynitrite. In particular, we studied the influence of CO2 on the rate and the mechanism of this reaction. We found that the peroxynitrite-mediated oxidation of MbFeO2 proceeds in two steps via the formation of MbFeIVdO also in the presence of CO2. However, the second-order rate constant for the first reaction step is significantly larger [(4.1 ( 0.7) × 105 M-1 s-1, at pH 7.5, 20 °C, and in the presence of 1.2 mM CO2]. In contrast, the rate constant for the peroxynitrite-mediated reduction of MbFeIVdO to metMb is only slightly larger in the presence of 1.2 mM CO2 [(3.2 ( 0.2) × 104 M-1 s-1, at pH 7.5 and 20 °C]. Finally, we show that because of the parallel decay of peroxynitrite and/or ONOOCO2-, under our experimental conditions, the oxidation of MbFeO2 to metMb by peroxynitrite is not a stoichiometric reaction. The amount of peroxynitrite required to completely oxidize MbFeO2 to metMb varies between 8 and 25 equiv, with larger amounts required to oxidize diluted MbFeO2 solutions in the presence of CO2.

Experimental Procedures Reagents. Buffer solutions were prepared from K2HPO4/ KH2PO4 (Fluka) with deionized Milli-Q water. Sodium dithionite and potassium superoxide were obtained from Fluka. Horse heart Mb was purchased from Sigma. Carbon dioxide was purchased from Carbagas (99.999%). Nitrogen monoxide was obtained from Linde and passed through a NaOH solution as well as a column of NaOH pellets to remove higher nitrogen oxides before use. Peroxynitrite, Carbon Dioxide, and Protein Solutions. Peroxynitrite was prepared from KO2 and gaseous nitrogen monoxide according to the literature (22) and stored in a polyethylene bottle or in small aliquots at -20 or at -80 °C. The peroxynitrite solutions contained variable amounts of nitrite (maximally 50% relative to the peroxynitrite concentration) and no hydrogen peroxide. The stock solution was diluted with 0.01 M NaOH and the concentration of the resulting solutions was determined spectrophotometrically prior to each experiment by measuring the absorbance at 302 nm [302 ) 1705 M-1 cm-1 (23)]. For the experiments carried out in the absence of added CO2, the buffers and the 0.01 M NaOH solutions were prepared fresh daily and thoroughly degassed. Experiments in the presence of CO2 were carried out by adding to the protein solution the required amount either from a saturated CO2 solution or from a freshly prepared 0.5 M sodium bicarbonate solution. The saturated CO2 solution (ca. 76 mM) was prepared by bubbling CO2 through water at 0 °C for at least 1 h. The values for the constant of the hydration-dehydration equilibrium CO2 + H2O h H+ + HCO3- were taken from ref 24, by taking into consideration the ionic strength of the solutions. After CO2 or bicarbonate was added, the protein solutions were allowed to equilibrate at room temperature for at least 5 min. The CO2 concentration is always expressed as the true concentration in equilibrium with HCO3-. Under our experimental conditions, the reaction of CO2 with ONOO- was much faster than the CO2 equilibration with HCO3-. Thus, the CO2 concentration during the reaction corresponded to the concentration in the protein solution, although the pH jumped to more alkaline values upon mixing with the peroxynitrite solution. MbFeO2 (oxyMb) was prepared by reducing metMb with a slight excess of sodium dithionite. The solution was purified chromatographically on a Sephadex G25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the eluent. In some

Chem. Res. Toxicol., Vol. 16, No. 3, 2003 391 cases, the protein was purified analogously a second time to ensure complete removal of sodium dithionite. However, this procedure proved not to be necessary, as it did not influence the results of the following experiments. The concentration of the MbFeO2 solutions was determined by measuring the absorbances at 417, 542, and/or 580 nm [417 ) 128 mM-1 cm-1, 542 ) 13.9 mM-1 cm-1, and 580 ) 14.4 mM-1 cm-1 (25)]. MbFeIVdO was prepared by adding 5 equiv of hydrogen peroxide to the MbFeO2 solution. The concentration of the MbFeIVdO solutions was determined by measuring the absorbance at 421 nm [421 ) 111 mM-1 cm-1 (26)]. Determination of the Amount of Peroxynitrite Required to Completely Convert MbFeO2 or MbFeIVdO to metMb. Absorption spectra were collected on a UVIKON 820, on an Analytik Jena Specord 200, or on an Analytik Jena Specord 100 spectrophotometer. The amount of peroxynitrite required to completely convert MbFeO2 or MbFeIVdO to metMb was determined in 0.1 M phosphate buffer, pH 7.4. For this purpose, peroxynitrite was either added as a bolus or titrated in the protein solution in the absence or presence of 1.2 mM CO2. In a typical experiment, the MbFeO2 solution (2 mL of a ca. 5-40 µM solution) was placed in a sealable cell, a peroxynitrite aliquot was added from a ca. 1 mM stock solution, and the cell was gently shaken. Finally, a spectrum was rapidly recorded (in ca. 20 s). If the reaction was not complete, an additional peroxynitrite aliquot was added immediately. The reaction was considered to be finished when addition of peroxynitrite did not induce further changes in the UV/vis spectrum. Stopped-Flow Kinetic Analysis. Kinetic studies were carried out with an Applied Photophysics SX17MV singlewavelength stopped-flow instrument and an On-Line Instrument Systems Inc. stopped-flow instrument equipped with an OLIS RSM 1000 rapid scanning monochromator. The width of the cells in the two spectrophotometers is 1 and 2 cm, respectively. In some of the experiments with the OLIS RSM apparatus, the width of the cell was also 1 cm. The mixing time of the instruments is 2-4 ms. If not specified, measurements were carried out at 20 °C. With the Applied Photophysics apparatus, kinetic traces were collected at different wavelengths between 300 and 650 nm and the data were analyzed with the SX17MV operating software or with Kaleidagraph, Versions 3.0.5 and 3.0.8. For the determination of the second-order rate constants, the traces were mostly collected at 589 and 612 nm. The results of the fits of the traces (averages of at least 10 single traces) from at least five experiments were averaged to obtain each observed rate constant, given with the corresponding standard deviation. Care was taken that the absolute absorbance of the reaction mixture was not higher than one absorbance unit. The pH was measured at the end of the reactions for control. The protein solutions were prepared by diluting MbFeO2 and MbFeIVdO stock solutions to the desired concentration with 0.1 M phosphate buffer (pH 6.0-7.5) under aerobic conditions. Peroxynitrite solutions were prepared by diluting the stock solution immediately before use with 0.01 M NaOH to achieve the required concentration. In general, the protein was dissolved in a 0.1 M buffer at a pH slightly more acidic than the desired final pH, which was always measured at the end of the reactions for control. Simulations. Kinetic simulations were carried out with the help of the Chemical Kinetic Simulator software, version 1.01 (from IBM, Almaden Research Center, http:// www.almaden.ibm.com/st/msim/) (27).

Results and Discussion Dependence of the Rate Constants for the Peroxynitrite-Mediated Oxidation of Mb on the Protein Concentration. We have recently shown that the peroxynitrite-mediated oxidation of MbFeO2 proceeds via an intermediate MbFeIVdO complex, which, in a second step, reacts further with peroxynitrite to yield metMb

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Figure 1. Dependence on the protein concentration of the observed pseudo-first-order rate constants for the two steps of the reaction between peroxynitrite (250 µM) and MbFeO2 (in 0.05 M phosphate buffer, pH 7.3, and 20 °C).

(21). In this previous work, we carried out all of the kinetic measurements with peroxynitrite in excess and at a constant protein concentration (about 2 µM) and determined the second-order rate constants for the two steps: (5.4 ( 0.2) × 104 and (2.2 ( 0.1) × 104 M-1 s-1, respectively (at pH 7.3 and 20 °C) (21). We have now investigated the influence of the protein concentration on the reaction rate. For this purpose, we have studied the reaction between different concentrations of MbFeO2 (0.5-22 µM) and an excess of peroxynitrite by single-wavelength stopped-flow spectroscopy. As in our previous studies (21), the two steps of the reactions were followed at 612 and 589 nm, the approximate isosbestic points for the second and the first reaction step, respectively. As shown in Figure 1, in the presence of a constant large excess of peroxynitrite (250 µM), the observed pseudo-first-order rate constants for the first step of its reaction with MbFeO2 increased with decreasing protein concentration (22-0.5 µM). This trend was more pronounced at very low protein concentrations. In contrast, the observed pseudo-first-order rate constants for the second step were nearly constant between 1 and 4 µM and slightly decreased at higher protein concentrations. In a narrower MbFeO2 concentration range (1-4.5 µM), we determined the second-order rate constants for the two reaction steps by varying the peroxynitrite concentration (50-450 µM). As shown in Figure 2 and summarized in Table 1, the second-order rate constants for the oxidation of MbFeO2 to MbFeIVdO, obtained from the linear fits of the plot of the observed pseudo-firstorder rate constants over peroxynitrite concentration (full symbols and lines), were invariant over this protein concentration range. However, the value of the y-axis intercept increased with decreasing protein concentration. The second-order rate constant for the second reaction step, the reduction of MbFeIVdO to metMb, was also constant within this protein concentration range (open symbols and dotted lines). The values of the intercepts of the four lines were nearly identical (within the error of the experiment). One possible explanation for these results is that in concentrated solutions Mb may aggregate and consequently partly hinder the reaction of peroxynitrite with the heme center. However, the concentration range

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Figure 2. Plots of kobs vs peroxynitrite concentration for the two steps (full symbols, MbFeO2 f MbFeIVdO; open symbols, MbFeIVdO f metMb) of the peroxynitrite-mediated oxidation of different concentrations of MbFeO2 (given in the legend) in 0.05 M phosphate buffer, pH 7.3, 20 °C. The second-order rate constants resulting from the linear fits depicted are summarized in Table 1. Table 1. Summary of the Second-Order Rate Constants (× 104 M-1 s-1) for the Peroxynitrite-Mediated Oxidation of Different Concentrations of MbFeO2 in 0.05 M Phosphate Buffer at pH 7.3 and 20 °C [MbFeO2] (µM)

MbFeO2 f MbFeIVdO

MbFeIVdO f metMb

1.1 2.1 3.2 4.5

4.8 ( 0.2 5.3 ( 0.2 3.8 ( 0.2 3.9 ( 0.2

1.85 ( 0.06 2.17 ( 0.02 1.85 ( 0.06 1.97 ( 0.02

within which significant changes are observed is not very large (1-4 µM) and studies in the presence of 0.5 M NaCl showed that the observed pseudo-first-order rate constants for the two steps of the reaction between MbFeO2 and peroxynitrite are not influenced by the ionic strength of the buffer (data not shown). Thus, other factors have to be responsible for these observations. Interestingly, the second-order rate constants for the reaction between MbFeO2 and peroxynitrite do not depend on the protein concentration (Figure 2). Thus, when determining the second-order rate constants, the MbFeO2 concentration has to be kept constant within a series. Nevertheless, the values of the second-order rate constants can be compared with those determined with other measurements carried out at different MbFeO2 concentrations. The rate of the reaction between peroxynitrite and iron(III) porphyrin complexes was also found to depend on the concentration of the complexes (28). In contrast to Mb, the values of the observed rate constants increased with increasing [FeIII(TMPyP)]5+ concentration (TMPyP ) meso-tetra(N-methyl-4-pyridyl)porphine). However, because of significant differences in the mechanisms of the reactions of peroxynitrite with Mb (21) and with the iron(III) porphyrin complexes (29), these trends were not expected to be identical. Amount of Peroxynitrite Required to Completely Convert MbFeO2 or MbFeIVdO to metMb. Under the conditions of our experiments, when peroxynitrite is mixed with a MbFeO2 solution, two parallel reactions always take place as follows: the decay of peroxynitrite and its reaction with the protein. Thus, more than 2 equiv is usually needed to completely oxidize MbFeO2 to


Figure 3. (A) UV/vis spectra of a MbFeO2 solution (7.5 µM in 0.1 M phosphate buffer, pH 7.0, dotted spectrum) after 48 subsequent additions of 0.15 equiv of peroxynitrite (5 µL of a 430 µM solution in 0.01 M NaOH) to yield metMb (bold spectrum). For clarity, each third spectrum is shown (each 0.45 equiv). (B) UV/vis spectra of a MbFeO2 solution (37.8 µM in 0.1 M phosphate buffer, pH 7.0, dotted spectrum) after five subsequent additions of 2 equiv of peroxynitrite (130 µL of a 1.1 mM solution in 0.01 M NaOH) to yield metMb (bold spectrum). All spectra were corrected for the dilution.

metMb. To find out whether the method used to add peroxynitrite had an influence on the amount required to completely convert MbFeO2 to metMb, we added peroxynitrite either as a bolus or in small fractions from a concentrated solution and followed the reaction by UV/ vis spectroscopy. As shown in Figure 3A, when a MbFeO2 solution (2 mL of a 7.5 µM solution in 0.1 M phosphate buffer pH 7.0) was titrated with subsequent additions of 0.15 equiv of peroxynitrite (5 µL of a ca. 430 µM solution in 0.01 M NaOH), the UV/vis spectrum of the reaction mixture showed the progressive transformation of MbFeO2 to metMb. The reaction was complete, which means that no further changes were observed in the UV/vis spectra, when approximately 7-8 equiv were added. When a MbFeO2 solution (2 mL of a 37.8 µM solution in 0.1 M phosphate buffer, pH 7.0) was titrated with subsequent additions of larger fractions of 2 equiv of peroxynitrite each (130 µL of a ca. 1.12 mM solution) under the same conditions, the reaction was complete after addition of 8-10 total equiv (Figure 3B). Finally, when peroxynitrite was added as a bolus, again about 8-9 equiv were needed to completely transform MbFeO2 to metMb (data not shown).

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To investigate whether the volumetric ratio of the peroxynitrite- and the MbFeO2-containing solutions influences the amount of peroxynitrite required to completely oxidize MbFeO2 to metMb under our experimental conditions, we mixed equal volumes of the two solutions by using a stopped-flow apparatus (OLIS RSM 1000). The reaction was followed by rapid-scan UV/vis spectroscopy. In a protein concentration range of 2.5-18.2 µM, also with this better mixing technique, approximately 8-9 equiv of peroxynitrite were required to completely convert MbFeO2 to metMb (data not shown). The amount of peroxynitrite needed to quantitatively reduce MbFeIVdO to metMb was determined analogously. When a MbFeIVdO solution (ca. 4 µM) was titrated with subsequent additions of aliquots of 1 equiv of peroxynitrite, the UV/vis spectrum of the reaction mixture showed the progressive transformation of MbFeIVdO to metMb. The reaction was complete when approximately 7 equiv were added. When peroxynitrite was added as a bolus, the reaction was complete after addition of ca. 8 equiv (data not shown). Interestingly, slightly lower amounts of peroxynitrite (ca. 6-7 equiv) were needed to completely convert a concentrated MbFeIVdO (ca. 30 µM) solution to metMb. Taken together, these results show that under our experimental conditions, because of the parallel decay of peroxynitrite, both the peroxynitrite-mediated oxidation of MbFeO2 to metMb and the reduction of MbFeIVdO to metMb are not stoichiometric processes. Within the MbFeO2 concentration range studied (4-30 µM), the amount of peroxynitrite needed to convert MbFeO2 to metMb does not depend significantly on the protein concentration. However, a slightly larger amount of peroxynitrite seems to be needed to convert MbFeO2 or MbFeIVdO to metMb when the peroxynitrite solution is added as a bolus, a result that suggests that under these conditions larger quantities of peroxynitrite decay without reacting with the protein. This result would imply that MbFeO2 and MbFeIVdO can scavenge slightly more efficiently peroxynitrite when it is titrated in the protein solution than when it is added as a bolus. Another possible explanation for this result is that when peroxynitrite is titrated in the protein solution, the experiment obviously lasts longer than when just one bolus is added. Thus, the slow reaction with nitrite (see below for details) may contribute to the oxidation of MbFeO2 and/or MbFeIVdO to metMb, resulting in an apparently lower amount of peroxynitrite required to quantitatively convert all of the protein to metMb. The local Mb concentration in vivo is significantly larger than that of peroxynitrite, which may often be generated continuously in very small concentrations (in the nanomolar range). Thus, our results indicate that under physiological conditions Mb may trap most peroxynitrite by its reaction with the heme center and thus prevent formation of reactive intermediates that may damage surrounding biomolecules. As the rate constant for the reaction of MbFeIVdO with peroxynitrite is only slightly lower than that for the peroxynitrite-mediated oxidation of MbFeO2 to MbFeIVdO, the strong oxidizing species MbFeIVdO does not accumulate in high concentrations, even when the protein is present in excess. For instance, analysis of the second spectrum of Figure 3A, measured after addition of three aliquots of 0.15 equiv of peroxynitrite each, indicates that ca. 8 and 7% of the protein are present as MbFeIVdO and metMb, respec-

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Figure 4. Dependence of the pseudo-first-order rate constants for the two steps (612 nm: open circles, MbFeO2 f MbFeIVdO; 589 nm: full circles, MbFeIVdO f metMb) of the reaction between peroxynitrite (275 µM) and MbFeO2 (2 µM) on the nitrite concentration, at pH 7.5 and 20 °C.

tively. This observation is important, as MbFeIVdO has been shown to promote oxidation, peroxidation (30-32), and epoxidation (33) of various biomolecules in vitro. In addition, •MbFeIVdO, the one electron oxidized form of MbFeIVdO, has been proposed to be responsible for oxidative damage in the ischemic and then reoxygenated heart (34). When determining the yield of the reaction between MbFeO2 and peroxynitrite, care must be taken to measure the UV/vis spectrum immediately after addition of peroxynitrite (within ca. 10-20 s) to avoid further reaction with nitrite, an ubiquitous contaminant of our peroxynitrite solutions. We found that addition of 1 equiv of peroxynitrite premixed with 3 equiv of nitrite (relative to the protein concentration) leads to the complete transformation of MbFeO2 to metMb within ca. 1.5 h. Nitrite is known to oxidize MbFeO2 to metMb at a significantly lower rate via an autocatalytic mechanism (35). Indeed, reaction of MbFeO2 with 3 equiv of nitrite leads to the conversion of only approximately 10% MbFeO2 to metMb in 1.5 h. In addition, nitrite is known to react with MbFeIVdO. The rate constant for this reaction has been measured as 16 ( 1 M-1 s-1 (at pH 7.5 and 20 °C) (36). Taken together, these data suggest that nitrite may cause the formation of metMb either directly, by reducing MbFeIVdO, or indirectly, by generating reactive species responsible for MbFeIVdO reduction. Influence of the Amount of Contaminating Nitrite on the Second-Order Rate Constants for the Peroxynitrite-Mediated Oxidation of MbFeO2. As mentioned above, as nitrite was always present in different concentrations as a contaminant of our peroxynitrite solutions, we determined its influence on the second-order rate constant of the reaction between MbFeO2 and peroxynitrite. For this purpose, we first determined by stopped-flow spectroscopy the observed pseudo-firstorder rate constants of the two steps of the reaction between MbFeO2 (2 µM) and peroxynitrite (275 µM) in the presence of increasing amounts of nitrite (150-600 µM). As shown in Figure 4, the values of the observed rate constants only slightly increased with increasing nitrite concentration. Moreover, we determined the secondorder rate constants of the two steps of the peroxynitrite-

Figure 5. Rapid scan UV/vis spectra of the reaction between MbFeO2 [(A) 4.7 µM and (B) 20 µM] and peroxynitrite [(A) 62 µM and (B) 210 µM] in the presence of CO2 (1.3 mM) in 0.05 M phosphate buffer at pH 7.5, 20 °C. Traces 1-11 were recorded in (A) 0, 4, 8, 12, 16, 36, 56, 76, 96, 116, and 136 ms after mixing and in (B) 0, 4, 8, 12, 16, 20, 32, 42, 52, 62, and 72 ms after mixing.

mediated oxidation of MbFeO2 in the presence of preadded nitrite, by varying the peroxynitrite concentration. In the presence of 150 and 600 µM nitrite, we obtained for the first step 3.6 × 104 and 3.5 × 104 M-1 s-1 and for the second step 1.8 × 104 and 1.7 × 104 M-1 s-1, respectively (at pH 7.5 and 20 °C, data not shown). Taken together, these data suggest that nitrite does not influence significantly the rate of the two reaction steps. Thus, these results confirm the hypothesis discussed above that the influence of nitrite on the yield of the reaction is due to an indirect reaction and not to the direct oxidation of MbFeO2 by nitrite. Influence of Carbon Dioxide on the Rate and on the Mechanism of the Peroxynitrite-Mediated Oxidation of MbFeO2. The rapid reaction between ONOOand CO2, which yields ONOOCO2- (5-7), represents one of the most significant routes for peroxynitrite consumption in biological systems. Here, we have carried out detailed mechanistic studies of the reaction between MbFeO2 and peroxynitrite in the presence of different amounts of CO2. As shown in Figure 5, in the presence of 1.3 mM CO2, the changes observed in the rapid-scan UV/vis spectra of the reaction between MbFeO2 and peroxynitrite were



Figure 6. Dependence of the pseudo-first-order rate constants for the two steps (612 nm: open circles, MbFeO2 f MbFeIVdO; 589 nm: full circles, MbFeIVdO f metMb) of the reaction between peroxynitrite (60 µM) and MbFeO2 (5 µM) on the CO2 concentration, at pH 7.4 and 20 °C.

qualitatively identical to those observed in the absence of CO2 (21). This observation suggests that addition of CO2 does not significantly alter the reaction mechanism. Thus, in the first step, MbFeO2 is oxidized to MbFeIVdO, which is then reduced to metMb. This conclusion is also confirmed by the presence of a set of isosbestic points at 588 and 524 nm for the first process, values very similar to those found in the absence of added CO2 (589 and 526 nm). Interestingly, for the second step of the reaction of MbFeO2 with peroxynitrite, no clean isosbestic points were detected in the presence of 1.3 mM CO2 (Figure 5B). In contrast, isosbestic points were found for both steps of the reaction in the region of the spectrum of the Soret band, at 411 and 414 nm in the presence of CO2 (Figure 5A) and at 413 and 417 nm in its absence (21) (for the first and second step, respectively). Finally, further evidence for the formation of MbFeIVdO was obtained by the identification, after addition of Na2S, of iron(II) sulfomyoglobin in an experiment analogous to that described for the reaction in the absence of CO2 (21). The kinetics of the reaction between MbFeO2 and peroxynitrite in the presence of CO2 were studied by single-wavelength stopped-flow spectroscopy under pseudofirst-order conditions with peroxynitrite in excess in the pH range of 6.1-8.3 and CO2 concentration range of 0-8 mM. As for the reactions in the absence of added CO2 (21), the first and the second steps of the reaction were followed at 612 and 589 nm, respectively. Despite the fact that no clear isosbestic point was observed at 612 nm, the absorbance changes arising at this wavelength from the second step of the reaction are negligible, and thus, the observed rate constant for the first reaction step can be determined. As shown in Figure 6, at pH 7.4, an excess of CO2 (0.2-2.4 mM) caused an apparently linear CO2 concentration-dependent increase of the observed pseudofirst-order rate constant for the peroxynitrite-mediated oxidation of MbFeO2 to MbFeIVdO. Analogously, the observed rate constant for the second step of the reaction, that is reduction of MbFeIVdO to metMb, also increased linearly with increasing CO2 concentration. As shown in Figure 7, at pH 7.5 and in the presence of different amounts of CO2 (0.2-2.4 mM) added either from a saturated CO2 solution (full symbols and lines) or from a bicarbonate stock solution (open symbols and dotted

Figure 7. Plots of kobs vs peroxynitrite concentration for the two steps (A, MbFeO2 f MbFeIVdO; B, MbFeIVdO f metMb) of the peroxynitrite-mediated oxidation of MbFeO2 (5 µM, pH 7.5, 20 °C) in the presence of different amounts of CO2 (given in the legend). For the experiments in the presence of 0.2-0.8 mM CO2 (full symbols and lines), CO2 was added from a saturated CO2 solution. The data in the presence of 0.8-2.5 mM CO2 (open symbols and dotted lines) were generated by adding the required amount of bicarbonate from a 0.5 M stock solution (see Experimental Procedures for details). The second-order rate constants resulting from the linear fits depicted are summarized in Table 2.

lines), the observed pseudo-first-order rate constants for the first reaction step were linearly dependent on the peroxynitrite concentration. As summarized in Table 2, the obtained second-order rate constants increased with increasing CO2 concentration (Figure 7A). In contrast, the observed pseudo-first-order rate constants for the second reaction step linearly increased with increasing peroxynitrite concentration only in the presence of low amounts of CO2 (0.2-0.8 mM). The second-order rate constants obtained for the reduction of MbFeIVdO to metMb in this CO2 concentration range were nearly constant (Table 2). In the presence of 0.8-2.4 mM CO2, the observed rate constants for the second reaction step were nearly constant in the peroxynitrite concentration range of 50-250 µM (Figure 7B). At higher CO2 concentrations, the observed rate constants even apparently decreased with increasing peroxynitrite concentration. As shown in Figure 7, the values of the y-axis intercepts of the linear fits increased with increasing concentration of added CO2.

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Table 2. Summary of the Second-Order Rate Constants (× 104 M-1 s-1) for the Peroxynitrite-Mediated Oxidation of MbFeO2 (5 µM) Measured at Different pH Values and 20 °C in the Presence of Different Amounts of CO2 [CO2] (mM) 0 0.2 0.4 0.8 1.2 1.6 2.4 3.6 7.3 a

MbFeO2 f MbFeIVdO

MbFeIVdOf metMb

pH 6.1

pH 6.8

pH 7.5

pH 8.3

pH 6.1

pH 6.8

pH 7.5

pH 8.3

4.4 ( 0.3

3.8 ( 0.3

4.4 ( 0.3 3.6 ( 0.3

44 ( 5 54 ( 2

1.9 ( 0.2 2.5 ( 0.6

1.4 ( 0.4 2.1 ( 0.6

2.2 ( 0.1 1.9 ( 0.1 1.9 ( 0.1 3.0 ( 0.2 3.2 ( 0.2 a a

1.2 ( 0.1

49 ( 2

3.2 ( 0.2 3.7 ( 0.1 3.5 ( 0.2 3.5 ( 0.4 2.7 ( 0.1 2.3 ( 0.3

17 ( 1 32 ( 3

5.4 ( 0.2 9.0 ( 0.5 15 ( 1 26 ( 2 41 ( 7 37 ( 4 53 ( 8

4.6 ( 0.2

9.8 ( 0.4 13 ( 2

3.5 ( 0.2 8.8 ( 0.6 9.6 ( 0.8 16 ( 2 18 ( 2 31 ( 3

1.5 ( 0.4

Negative slope.

The pH-independent second-order rate constant for the reaction of ONOO- with CO2 has been determined as 3 × 104 M-1 s-1 at 24 °C (6) and 6 × 104 M-1 s-1 at 37 °C (5). Under the conditions of our experiments, the halflife of peroxynitrite is approximately 46 ms in the presence of 0.5 mM CO2 and about 3 ms in the presence of 7.3 mM CO2. Consequently, our results suggest that MbFeO2 reacts more rapidly with the product(s) of the reaction between CO2 and ONOO- than with HOONO. Saturation of the curve in Figure 8A can be explained as follows: when peroxynitrite is mixed with protein solutions containing amounts of CO2 in the lower concentration range, both peroxynitrite and ONOOCO2(and/or the products derived from its decay) are present at the beginning of the reaction, after the 2-4 ms needed to mix the two solutions with the stopped-flow apparatus. In contrast, in the presence of CO2 concentrations higher than ca. 8 mM, within the mixing time, peroxynitrite is completely converted to the adduct ONOOCO2- (and/or the products derived from its decay). Thus, at lower CO2 concentrations, the observed rate constant is smaller because of the concurrent (slower) direct reaction of peroxynitrite with MbFeO2. In principle, under these conditions, both peroxynitrite and ONOOCO2- (and/or the products derived from its decay) may react with MbFeO2. Taken together, our results suggest that the saturation value corresponds to the effective value of the reaction of MbFeO2 with ONOOCO2- (and/or the products derived from its decay). However, as the reaction between peroxynitrite and CO2 is reversible (37), despite the large rate constant for the forward reaction, a small fraction of peroxynitrite will always be present at equilibrium and, possibly, react directly with the protein. Figure 8. Dependence of the second-order rate constants for the two steps (A, MbFeO2 f MbFeIVdO; B, MbFeIVdO f metMb) of the peroxynitrite-mediated oxidation of MbFeO2 (5 µM) on the CO2 concentration, at pH 6.8 and 20 °C.

As shown in Figure 8 and summarized in Table 2, a similar trend was observed when the reaction was studied at pH 6.8 over a large CO2 concentration range (0.2-7.3 mM). The second-order rate constants for the first step of the peroxynitrite-mediated oxidation of MbFeO2 increased with increasing CO2 concentrations up to ca. 7.3 mM CO2 [(5.4 ( 0.2) × 105 M-1 s-1]. At this concentration, a saturation effect was observed. In contrast, the values of the second-order rate constants for the second step were all in the range of (1.4-3.7) × 104 M-1 s-1 and apparently decreased with increasing CO2 concentration.

As the half-life of the adduct generated from the reaction of ONOO- with CO2 is very short [0.5-3 ms (7, 11, 37)], the second step of the reaction between MbFeO2 and peroxynitrite in the presence of CO2 must consist of the reduction of MbFeIVdO to metMb byproducts derived from the decay of ONOOCO2- (see discussion of simulations below). The adduct ONOOCO2- has been proposed to partly decompose to NO2• and CO3•-, although the yield of free radical formation is still a matter of discussion (10, 11). Thus, NO2• is likely to be the species responsible for MbFeIVdO reduction. Indeed, this reaction has been shown to rapidly (ca. 107 M-1 s-1) generate metMb and nitrate (38). As shown in Figure 9, in both the absence and the presence of 1.3 mM CO2, the values of the second-order rate constants of the reactions between MbFeIVdO and peroxynitrite are nearly identical to those of the second



Figure 9. Plots of kobs vs peroxynitrite concentration for the second step (MbFeIVdO f metMb) of the peroxynitrite-mediated oxidation of MbFeO2 (5 µM) in the absence and in the presence of 1.3 mM CO2 at pH 6.8 and 20 °C. Comparison with the data obtained from the reaction of separately prepared MbFeIVdO and peroxynitrite under the same conditions.

steps of the reactions between peroxynitrite and MbFeO2 under the same conditions. This result again confirms the hypothesis that the second step of the reaction of MbFeO2 with peroxynitrite in the presence of CO2 is not due to interactions of MbFeIVdO with products derived from the first step of the reaction. pH Dependence of the Reaction Rate in the Presence of Carbon Dioxide. As shown in Figure 10 and summarized in Table 2, at a given CO2 concentration (1.2 mM), the second-order rate constant of the first reaction step, the oxidation of MbFeO2 to MbFeIVdO by peroxynitrite, increased with increasing pH. This observation supports the mechanistic hypothesis that MbFeO2 reacts rapidly with the product(s) of the reaction between CO2 and ONOO-, which are formed at a larger extent under alkaline conditions. Interestingly, the value of the second-order rate constant of the reaction between MbFeO2 and peroxynitrite obtained at pH 6.8 in the presence of 7.3 mM CO2, (5.4 ( 0.2) × 105 M-1 s-1, is almost identical to that measured at pH 8.3 in the presence of 1.2 mM CO2, (4.9 ( 0.2) × 105 M-1 s-1. As discussed above, in the presence of 1.2 mM CO2, not all peroxynitrite will have reacted with CO2 within the mixing time of the stopped-flow apparatus. However, the amount of ONOOCO2- generated in the mixing time at pH 8.3 is larger than that formed at pH 6.8, as the concentration of ONOO- is larger under alkaline conditions. Thus, these two effects may compensate each other and the second-order rate constants at different pH values and CO2 concentrations may be similar. Interestingly, the second-order rate constants for the second step of the reaction of MbFeO2 and peroxynitrite in the presence of 1.2 mM CO2 did not show a clear pH dependence. The values between pH 6.1 and pH 7.5 are nearly identical whereas that at pH 8.3 is slightly smaller. However, at a given peroxynitrite concentration, the observed rate constants mostly increased with increasing pH. This result supports the hypothesis discussed above that the second step corresponds to the reduction of MbFeIVdO by NO2•. Under basic conditions,

Figure 10. Plots of kobs vs peroxynitrite concentration for the two steps (A, MbFeO2 f MbFeIVdO; B, MbFeIVdO f metMb) of the peroxynitrite-mediated oxidation of MbFeO2 (5 µM) in the presence of 1.2 mM CO2 at different pH values and 20 °C. The second-order rate constants resulting from the linear fits depicted are summarized in Table 2.

larger amounts of peroxynitrite will decay through the reaction with CO2 and thus generate larger amounts of NO2•. When larger amounts of NO2• are generated, the concurring dimerization of NO2• to N2O4 with subsequent hydrolysis to nitrite and nitrate may become more significant. Thus, the observed reaction rate may not increase significantly with increasing peroxynitrite concentration. Several factors may influence the reaction rate in opposite ways in the presence of CO2. At lower pH, it has been shown that the distal histidine is protonated and swings out of the heme pocket (39). Thus, the direct reaction with the iron center may be facilitated. However, at lower pH, the concentration of the anion ONOO-, the species that reacts with CO2, is lower. Amount of Peroxynitrite Required to Completely Convert MbFeO2 or MbFeIVdO to metMb in the Presence of CO2. Analogously to the experiments described above in the absence of CO2, we determined the amount of peroxynitrite required to completely oxidize MbFeO2 to metMb at pH 7.2 in the presence of 1.2 mM CO2. Peroxynitrite was added from a concentrated stock solution either as a bolus or in small aliquots to the protein solution, and the reaction was followed by UV/vis spectroscopy. In addition, to exclude artifacts due

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to inhomogeneous mixing of different volumes, we also mixed equal volumes of the two solutions by using a stopped-flow apparatus (OLIS RSM 1000). As in the absence of added CO2, the results of these different mixing techniques were nearly identical. Interestingly, we found that the amount of peroxynitrite needed to completely oxidize MbFeO2 in the presence of CO2 depends on the protein concentration. When diluted MbFeO2 solutions (ca. 4 µM) were mixed with peroxynitrite, approximately 25 equiv were needed to completely convert it to metMb. In contrast, only 10 equiv were needed to quantitatively oxidize concentrated MbFeO2 solutions (ca. 30 µM) (data not shown). The amount of peroxynitrite necessary to quantitatively reduce MbFeIVdO to metMb in the presence of 1.2 mM CO2 was determined analogously. Also in this case, a difference was noticed between diluted and concentrated solutions. When a ca. 4 µM MbFeIVdO solution was titrated by addition of small aliquots of peroxynitrite, the reaction was complete after approximately 15 equiv. In contrast, only about 6-8 equiv of peroxynitrite were needed to completely convert a concentrated MbFeIVdO (ca. 30 µM) solution to metMb. The large difference between the amount of peroxynitrite required to completely oxidize diluted vs concentrated MbFeO2 solutions in the presence of 1.2 mM CO2 may be explained by considering all of the reactions that can take place in the system. Obviously, addition of 1 equiv of peroxynitrite to a diluted (4 µM) or a concentrated (30 µM) MbFeO2 solution leads to different absolute peroxynitrite concentrations in the reaction mixtures (4 vs 30 µM). Therefore, in the experiments with larger peroxynitrite concentrations, MbFeO2 competes efficiently with CO2 and its direct oxidation by peroxynitrous acid becomes a major reaction pathway. The pseudo-firstorder rate constant of the reaction of peroxynitrite anion with CO2 does not depend on the peroxynitrite concentration as CO2 is in large excess (pseudo-first-order conditions) and is consequently identical in the experiments with low and high protein concentrations. Thus, in the presence of larger protein (and peroxynitrite) concentrations, the absolute amount of peroxynitrite required to transform MbFeO2 into metMb is lower. The observation that larger amounts of peroxynitrite are required to completely convert MbFeO2 to metMb in the presence of 1.2 mM CO2 (as compared to the amount required when no CO2 is added) can be explained by the significantly faster decomposition rate of ONOOCO2- vs HOONO. Thus, in the presence of CO2, a larger amount of peroxynitrite rapidly decomposes without interacting with the protein. Simulations. To get a better understanding of the mechanism of the reaction between MbFeO2 and peroxynitrite, in both the absence and the presence of added CO2, kinetic simulations were performed with the Chemical Kinetic Simulator software (27). The obtained concentration vs time profiles were compared to the original data. In some cases, kinetic traces were calculated for the absorbance changes at 589 and 612 nm, by using the extinction coefficients of the three protein forms involved. The reaction of peroxynitrite with MbFeO2 was initially modeled by using the following reactions 1-4, with the corresponding rate constants (at 20-25 °C):

Figure 11. Simulation of the reaction of 53 µM peroxynitrite with 2 µM MbFeO2 in the absence (A) and in the presence (B) of 1.2 mM CO2 at pH 7.0. (A) Thin lines, open symbols: simulation with equations 1-4; thick lines, full symbols: simulation with equations 1, 2a,b, and 3-7. (B) Thin lines, open symbols: simulation with equations 1-4 and 12-15; thick lines, full symbols: simulation with equations 1, 2a,b, 3-7, 12, 13a′,b′, and 14-16.

ONOO- a HOONO

k1 ) k-1 ) 104 s-1

(1)

HOONO f NO3- + H+ k2 ) 1.2 s-1 (40)

(2)

MbFeO2 + HOONO f MbFeIV ) O + NO2- + O2 + H+ k3 ) 16 × 104 M-1 s-1 (21) (3) MbFeIV ) O + HOONO + H+ f metMb + ONOO• k4 ) 6 × 104 M-1 s-1 (21) (4) The reaction was first simulated at pH 7.0. For this purpose, the rate constants for the protonation (k1) and the deprotonation (k-1) of peroxynitrite were fixed to an equal value (104 s-1). Larger (but of course equivalent) values for the two rate constants did not change the outcome of the simulations but led to longer calculation times. As we previously assumed that peroxynitrous acid is the only species that reacts with both MbFeO2 and MbFeIVdO (21), the pH-independent second-order rate constants for reactions 3 and 4 were set at twice the values measured at pH 7.0. As shown in Figure 11A (thin



lines), simulation of the reaction of 53 µM peroxynitrite with 2 µM MbFeO2 by using reactions 1-4 did not lead to the complete conversion of the protein to metMb. Indeed, approximately 15% of MbFeIVdO was still present after 5 s of reaction time. To get a better simulation of the experimental data, we had to consider the possibility that about 30% of HOONO decays to HO• and NO2• (41-43). Nitrogen dioxide has been shown to rapidly reduce MbFeIVdO to metMb (38). Thus, this reaction was also included in the simulation (eq 5). Obviously, also, the concurring dimerization of NO2• to N2O4, with its subsequent hydrolysis to nitrite and nitrate, had to be considered (eqs 6 and 7). As shown in Figure 11A (thick lines), simulation of the reaction between 53 µM peroxynitrite and 2 µM MbFeO2 with eqs 1, 2a,b, and 3-7 led to the complete conversion of MbFeO2 to metMb.

radical mechanism, only about 30% of MbFeO2 was oxidized by 53 µM peroxynitrite. Thus, the rate of dissociation of O2 from MbFeO2 is too slow to allow for the reaction to proceed exclusively via deoxyMb. In conclusion, it is conceivable that the reaction of MbFeO2 with peroxynitrite proceeds both via the direct oxidation of MbFeO2 and the reaction with deoxyMb.

HOONO f NO3- + H+

The reaction between 53 µM peroxynitrite and 2 µM MbFeO2 was also simulated at pH 8.0. The pH was set by defining the rate constant for the protonation (k1 ) 104 s-1) to be 10 times lower than that for the deprotonation (k-1 ) 105 s-1). The results of the simulations with the two models (radical/no-radicals) were identical to those discussed above for the reactions at pH 7.0. A mechanism involving eqs 1, 2a,b, and 3-7 was required to completely oxidize MbFeO2 with low peroxynitrite concentrations, whereas no difference between the two models was observed for the simulation of the reaction of MbFeO2 with larger peroxynitrite concentrations. In addition, we found that the simulated reaction at pH 8.0 proceeded too slowly, if it was assumed that peroxynitrous acid was the only species that reacted with the protein. However, the experimental data could be simulated sufficiently well with the assumption that MbFeO2 and MbFeIVdO reacted also with the anionic form of peroxynitrite. A good simulation was obtained by assuming that the rate constants for the reactions of MbFeO2 and MbFeIVdO with ONOO- are 10 times lower than those for the reactions with the protonated form (eqs 10 and 11). Addition of eqs 10 and 11 to the scheme used above to model the reaction at pH 7.0 did not influence the results of that simulation.

k2a ) 0.8 s-1 (44)

(2a)

HOONO f NO2• + HO• k2b ) 0.4 s-1 (44)

(2b)

MbFeIV ) O + NO2• f metMb + NO3k5 ) 1 × 107 M-1 s-1 (38) 2NO2• a N2O4

(5)

k6 ) 4.5 × 108 M-1 s-1 (45) (6) k-6 ) 6.9 × 103 s-1 (45)

N2O4 + H2O f NO2- + NO3- + 2H+ k7 ) 1 × 103 s-1 (45)

(7)

The second step of the reaction, that is, the reduction of MbFeIVdO to metMb, was also simulated independently. As for the simulation of the complete reaction, the contribution of the radicals was essential to entirely reduce 2 µM MbFeIVdO with 53 µM peroxynitrite. However, with eqs 1, 2a,b, and 4-7, the simulated reaction proceeded too rapidly. A better simulation of the experimental data could be obtained by either lowering the value of the rate constant of reaction 4 to 1 × 104 M-1 s-1 and/or by considering reaction 5 the only possible way to reduce MbFeIVdO to metMb. Interestingly, the results of the simulation of the reaction of MbFeO2 with higher peroxynitrite concentrations were nearly identical when either of the two different models discussed above (radicals/no-radicals) was used. Indeed, simulation of the reaction between 200 µM peroxynitrite and 2 µM MbFeO2 with either eqs 1-4 or eqs 1, 2a,b, and 3-7 led in both cases to the complete conversion of MbFeO2 to metMb. We have previously shown that the reaction of deoxyMb with peroxynitrite is significantly faster than that with MbFeO2 (k ≈ 106 M-1 s-1) (21). Thus, in our previous work, we suggested that the reaction of MbFeO2 with peroxynitrite may proceed exclusively via deoxyMb, after dissociation of dioxygen (reactions 8 and 9). Simulation of this reaction scheme, performed by using reactions 1-4, 8, and 9, showed that the proposed mechanism is not correct. Indeed, by using both the radical or the no-

MbFeO2 a MbFeII + O2 k8 ) 2 × 107 M-1 s-1 (46)

(8)

k-8 ) 24 s-1 (46) MbFeII + HOONO f MbFeIV ) O + NO2k9 ) 2 × 106 M-1 s-1 (21)

(9)

MbFeO2 + ONOO- f MbFeIV ) O + NO2- + O2 k10 ) 16 × 103 M-1 s-1 (this work) (10) MbFeIV ) O + ONOO- f metMb + ONOO• k11 ) 6 × 103 M-1 s-1 (this work)

(11)

Finally, we also attempted to simulate the reaction of MbFeO2 with peroxynitrite in the presence of 1.2 mM CO2. As shown in Figure 11B, the experimental data could not be simulated by assuming a direct reaction between the peroxynitrite/CO2 adduct (ONOOCO2-) and the protein. Indeed, when eqs 1-4 and 12-15 were used to simulate the reaction of 53 µM peroxynitrite with 2 µM MbFeO2, only very low amounts of MbFeO2 were oxidized to MbFeIVdO, and no metMb was formed (Figure 11B, thin lines). At higher peroxynitrite concentrations (400 µM), the result of the simulation was comparable: no complete oxidation of MbFeO2 was obtained and MbFeIVdO was always generated at a concentration higher than that of metMb.

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Herold et al.

ONOO- + CO2 f ONOOCO2k12 ) 3 × 104 M-1 s-1 (6) (12) ONOOCO2- f NO3- + CO2 k13 ) 500 s-1 (7)

(13)

MbFeO2 + ONOOCO2- f MbFeIVdO + ... k14 ) 6 × 105 M-1 s-1

(14)

MbFeIVdO + ONOOCO2- f metMb + ... k15 ) 6 × 104 M-1 s-1

(15)

Thus, also for the simulation of the reaction between MbFeO2 and peroxynitrite in the presence of 1.2 mM CO2, the assumption was made that radicals are involved in the reaction. Indeed, it has been proposed that 30% (11, 48) or less than 5% (10) of ONOOCO2- decays to form the free radicals NO2• and CO3•- (eqs 13a,b). However, also the simulation with eqs 1, 2a,b, 3-7, 12, 13a′,b′, 14, and 15 did not lead to complete oxidation of 2 µM MbFeO2 with 53 µM peroxynitrite. Nevertheless, this simulation was closer to the experimental data than that performed without considering the reactions with the radicals. In particular, the concentration of metMb obtained was larger than that of MbFeIVdO (data not shown). In addition, with this reaction scheme, simulation of the reaction of larger amounts of peroxynitrite (400 µM) with MbFeO2 (2 µM) led to almost complete formation of metMb. However, the rate of the simulated reaction was significantly slower than that measured experimentally under the same conditions. Interestingly, only small changes were observed when the simulations were carried out by considering the lower radical yields (reactions 13a′′ and 13b′′). Specifically, when it was assumed that only 5% free radicals was formed from ONOOCO2-, larger amounts of MbFeIVdO remained at the end of the simulation of the reaction.

ONOOCO2- f NO3- + CO2 k13a′ ) 350 s-1 (11, 48) k13a′′ ) 475 s-1 (10)

(13a)

ONOOCO2- f NO2• + CO3•k13b′ ) 150 s-1 (11, 48) k13b′′ ) 25 s-1 (10)

(13b)

Attempts were made to get a better simulation of the experimental data by introducing an additional possible reaction. The radical CO3•- is a strong oxidant [Eο ) 1.59 V (49)]. Therefore, it is conceivable that it can also oxidize MbFeO2 to metMb (eq 16). The rate constant for this reaction was estimated to be k ≈ 106 M-1 s-1. As shown in Figure 11B (thick lines), simulation of the reaction of 53 µM peroxynitrite with 2 µM MbFeO2 (in the presence of 1.2 mM CO2) by using eqs 1, 2a,b, 3-7, 12, 13a′,b′, and 14-16 led to a higher conversion of MbFeO2 to metMb. However, the rate of the second step of the simulated reaction was significantly faster than that determined experimentally. Interestingly, omitting the two direct reactions between ONOOCO2- and the protein

(eqs 14 and 15) did not influence the result of the simulation.

MbFeO2 + CO3•- f metMb + ... k16 ) 1 × 106 M-1 s-1 (16) It has recently been proposed that the reactive species ONOOCO2- is extremely short-lived and decomposes before it can react with biological molecules (37). In particular, it has been suggested that ONOOCO2- rapidly decays to NO2• and CO3•-, which then forms NO3- and CO2 (eqs 12a, 13c, and 17).

ONOO- + CO2 a ONOOCO2k12 ) 3 × 104 M-1 s-1 (6) k-12 ) 1.5 × 106 s-1 (37)

(12a)

ONOOCO2- a NO2• + CO3•k13c ) 1.9 × 109 s-1 (37) k-13c ) 5 × 108 M-1 s-1 (37)

(13c)

NO2• + CO3•- f NO3- + CO2 k17 ) 5 × 108 M-1 s-1 (47)

(17)

A final attempt was thus made to use this set of equations to simulate our system. The reaction of 53 µM peroxynitrite and 2 µM MbFeO2 in the presence of 1.2 mM CO2 was thus simulated with the eqs 1, 2a,b, 3-7, 12a, 13c, 16, and 17. Also with this reaction scheme, the experimental data could not be fitted satisfactorily. Oxidation of MbFeO2 was not complete and significant amounts of MbFeIVdO were present at the end of the simulated reaction. The simulation could be qualitatively improved by increasing the rate constant for reaction 16 to k16 ) 107 M-1 s-1. Under these conditions, MbFeO2 was completely oxidized to metMb, but the rate of the reaction was significantly faster than that measured experimentally. Mechanistic Considerations and Biological Significance. Despite the fact that we were not able to entirely reproduce the experimental data, a few conclusions can be drawn from the simulations described above. First, in the absence of CO2, we found that MbFeO2 must react also with ONOO- in order to explain the secondorder rate constants measured under basic conditions. The rate constant for the reaction of MbFeO2 with the anionic form of peroxynitrite was estimated as 1.6 × 104 M-1 s-1, that is, approximately 10 times lower than that of the reaction with HOONO. In addition, we found that the dissociation of dioxygen from MbFeO2 is too slow to allow for the peroxynitrite-mediated oxidation of MbFeO2 to proceed exclusively via the oxidation of deoxyMb (as erroneously suggested in ref 21). Finally, to get a complete oxidation of MbFeO2 to metMb with low amounts of peroxynitrite, MbFeIVdO must react not only with peroxynitrite but also (or exclusively) with NO2•. This observation may explain the requirement of several equivalents of peroxynitrite to completely reduce MbFeIVdO to metMb. Not only the isomerization of peroxynitrous acid but also the dimerization of NO2• to N2O4 and its subsequent hydrolysis to nitrite and nitrate compete with the reaction of NO2• with the protein. Thus, under our


experimental conditions, about 6-7 equiv of peroxynitrite are required to get a quantitative reduction of MbFeIVdO to metMb. The reaction in the presence of CO2 is more difficult to understand. The observation that the linear fits of the plots of the observed rate constants vs peroxynitrite concentration do not cross the origin and that the value of the intercept depends on the CO2 concentration implies a complex mechanism. From our simulations described above, it appears that in order to get complete conversion of MbFeO2 to metMb reactions with CO3•- and NO2• may have to be taken into account. The radical CO3•- is a strong oxidant. Thus, it is conceivable that it can oxidize MbFeO2 to metMb, and possibly also metMb to MbFeIVdO. The former reaction has been included in our simulation schemes, with an estimated bimolecular rate constant of 106-107 M-1 s-1. Preliminary experiments showed that the rate constant for the reduction of MbFeIVdO by NO2• is ca. 107 M-1 s-1 (38). This reaction was also included in our simulations. However, because of the uncertainties linked with these estimated rate constants, it is impossible to get a better simulation of the experimental data. In conclusion, we have shown that MbFeO2 reacts rapidly with peroxynitrite also in the presence of physiological amounts of CO2. Despite the significantly larger value of the rate constant for this reaction in the presence of CO2, MbFeO2 is less efficient in scavenging peroxynitrite in the presence of CO2 because larger amounts of peroxynitrite are needed to generate quantitatively metMb. Thus, only a small amount of peroxynitrite decomposes through reactions with the heme. The data presented in this work are in good agreement with our recent results, which show that MbFeO2 is nitrated by peroxynitrite to a significantly larger extent in the presence of added CO2 (50). In addition, the kinetic data presented here explain our observation that MbFeO2 protects free tyrosine less efficiently from peroxynitritemediated nitration in the presence of physiological amounts of CO2 (50). The mechanisms discussed in this work for the reaction of MbFeO2 with peroxynitrite in the presence of CO2 suggest that Mb probably does not react directly with ONOOCO2- (as proposed in ref 21) but scavenges the radicals derived from its decomposition. Thus, to get a better understanding of the mechanism of this reaction, the rate constants of the reactions of MbFeO2, metMb, and MbFeIVdO with CO3•- and NO2• have to be determined. Experiments are in progress in our laboratory to try to measure them.

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TX025595L

The Mechanism of the Peroxynitrite-Mediated Oxidation of Myoglobin

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