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Kinetic and Mechanistic Studies of the Peroxynitrite-Mediated Oxidation of Oxymyoglobin and Oxyhemoglobin Michael Exner and Susanna Herold* Laboratorium fu¨ r Anorganische Chemie, Eidgeno¨ ssische Technische Hochschule, Universita¨ tsstrasse 6, CH-8092 Zu¨ rich, Switzerland Received December 13, 1999
Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin (MbFeO2) and oxyhemoglobin (HbFeO2) showed that the mechanisms of these reactions are more complex than what had previously been reported; both reactions proceed in two steps. For myoglobin, we found that the small amount of deoxymyoglobin (MbFeII) which is in equilibrium with MbFeO2 is first oxidized by peroxynitrous acid to ferryl myoglobin (MbFeIVdO). Then, in the second step, MbFeIVdO is reduced by peroxynitrous acid to metmyoglobin (metMb). The secondorder rate constant values obtained at pH 7.3 and 20 °C for the two steps are (5.4 ( 0.2) × 104 and (2.2 ( 0.1) × 104 M-1 s-1, respectively. Analogous studies with hemoglobin suggest that its reaction with peroxynitrite follows the same mechanism. In this case, the second-order rate constant values measured at pH 7.0 and 20 °C for the two steps are (8.8 ( 0.4) × 104 and (9.4 ( 0.7) × 104 M-1 s-1, respectively. A possible mechanism in the absence as well as in the presence of CO2 and the relevance of these reactions in vivo are discussed.
Introduction Peroxynitrite1 is a strong oxidant that can be formed in vivo by the nearly diffusion-controlled reaction of nitrogen monoxide and superoxide (1, 2). Peroxynitrite anion (ONOO-) is stable, but its protonated form, peroxynitrous acid [HOONO, pKa ) 6.8 (3)], isomerizes to nitrate with a rate constant of 1.2 s-1 at 25 °C (3). The chemistry of peroxynitrite is currently being actively investigated (4). One- or two-electron oxidations by peroxynitrite can damage DNA (5), initiate lipid peroxidation (6), or modify aromatic- or sulfur-containing amino acid residues (7-9). Peroxynitrite nitrates aromatic compounds in a reaction that can be catalyzed by metal complexes (10) or metal-containing proteins (10-12). Moreover, ONOO- reacts rapidly with CO2 to yield 1-carboxylato-2-nitrosodioxidane (ONOOCO2-) (13-15), a stronger nitrating agent than peroxynitrite (16, 17). When the typical concentrations of different biological targets and their relative rates of reaction with peroxynitrite are taken into consideration, it appears that in vivo peroxynitrite should mainly disappear by reacting with carbon dioxide, glutathione, selenium-containing proteins, or metalloproteins, in particular heme proteins (1, 18-21). Thus, one of the targets for peroxynitrite in the blood vessels is hemoglobin. It has been reported that peroxynitrite reacts with oxyhemoglobin (HbFeO2)2 to yield methemoglobin (14, 22) and that this reaction is * To whom correspondence should be addressed: Laboratorium fu¨r Anorganische Chemie, Universita¨tsstrasse 6, ETH-Zentrum, CH-8092 Zu¨rich, Switzerland. E-mail:
[email protected]. Fax: (41) (1) 632 10 90. 1 The recommended IUPAC nomenclature for peroxynitrite is oxoperoxonitrate(1-); for peroxynitrous acid, 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).
likely to take place in vivo as peroxynitrite is able to diffuse across the red blood cell membrane (23, 24). During the course of this study, two groups have determined a value of the second-order rate constant for this reaction at pH 7.4, that is, 1 × 104 M-1 s-1 at 25 °C (24) and 2.5 × 104 M-1 s-1 at 37 °C (25). In a recent ESR study, it was shown that, in the presence of an excess of CO2, peroxynitrite reacts either with oxyhemoglobin or with human erythrocytes to yield a tyrosyl-centered radical (26). The reaction has been proposed to proceed via the ferryl species HbFeIVdO (26). Here we present a detailed kinetic study of the peroxynitrite-mediated oxidations of oxymyoglobin (MbFeO2) and oxyhemoglobin (HbFeO2). Our results3 (27) show that the mechanisms of these reactions are more complex than what had previously been reported and that the ferryl forms of the proteins, MbFeIVdO and HbFeIVdO, are involved. The data obtained by varying the pH and the oxygen concentrations indicate that deoxymyoglobin and peroxynitrous acid are likely to be the true reactants in this system.
Experimental Procedures Reagents. Buffer solutions were prepared from K2HPO4/KH2PO4 (Fluka) with deionized Milli-Q water. Peroxynitrite was prepared according to the method described in ref 28 and stored in a polyethylene bottle at -20 °C. The concentration of the solution was determined by measuring the absorbance at 302 2 Abbreviations: ESR, electron spin resonance spectroscopy; Hb, hemoglobin; Mb, myoglobin; HbFeO2, oxyhemoglobin; MbFeO2, oxymyoglobin; metHb, iron(III) hemoglobin; metMb, iron(III) myoglobin; MbFeIVdO, ferryl myoglobin; HbFeIVdO, ferryl hemoglobin; MbFeII, deoxymyoglobin; HbFeII, deoxyhemoglobin. 3 These results were presented in part at the Second International Conference on the Biology and Chemistry of Peroxynitrite, Heraklion, Greece, May 1999.
10.1021/tx990201k CCC: $19.00 © 2000 American Chemical Society Published on Web 03/28/2000
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nm (302 ) 1705 M-1 cm-1) (29). Absorption spectra were collected on a UVIKON 820 spectrophotometer. Deoxymyoglobin solutions were prepared by reducing lyophilized horse heart myoglobin obtained from Sigma with a slight excess of sodium dithionite (Fluka). The solution was purified chromatographically under argon on a Sephadex G25 column by using a degassed 0.1 M phosphate buffer solution (pH 7) as the eluent. If necessary, the solution was diluted in gastight SampleLock Hamilton syringes. Oxymyoglobin solutions were prepared analogously but were allowed to equilibrate with air during chromatographic purification. Ferryl myoglobin was prepared by adding 5 equiv of hydrogen peroxide to the oxymyoglobin solution. The concentrations of the oxy-, deoxy-, and ferryl myoglobin solutions were determined by measuring the absorbance at 417 nm (417 ) 128 mM-1 cm-1), 435 nm (435 ) 121 mM-1 cm-1) (30), and 421 nm (421 ) 111 mM-1 cm-1) (31), respectively. Pure human oxyhemoglobin stock solution HbA0 (57 mg/mL solution with approximately 1.1% methemoglobin) was a kind gift from APEX Bioscience, Inc. Oxyhemoglobin solutions were prepared by diluting the stock solution with buffer, and concentrations were determined by measuring the absorbance at 415, 541, and/or 577 nm (415 ) 125 mM-1 cm-1, 541 ) 13.8 mM-1 cm-1, and 577 ) 14.6 mM-1 cm-1) (30). Carbon dioxide solutions were prepared by saturating water for at least 1 h with CO2 at 0 °C. The obtained stock solution (ca. 78 mM) was diluted with degassed buffer to the required concentration. Stopped-Flow Kinetic Analysis. Kinetic studies were carried out with an Applied Photophysics SX17MV singlewavelength stopped-flow instrument, and with an On-Line Instrument Systems 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. The data collected with the Applied Photophysics apparatus were analyzed with the SX17MV operating software, with Microcal’s Origin 5.0, or with Kaleidagraph, version 3.0.5. Traces (averages of at least 10 single traces) from at least five experiments were averaged to obtain each observed rate constant. The protein solutions, in 0.1 M phosphate buffer, were mixed with the peroxynitrite solution, in 0.01 M NaOH, and the pH was always measured at the end of the reactions.
Exner and Herold
Figure 1. Rapid-scan UV-vis spectra of the reaction between 15 µM MbFeO2 and 195 µM peroxynitrite in 0.05 M phosphate buffer at pH 6.7 and 20 °C. For the first step of the reaction (A, 1-128 ms), traces are shown every 32 ms. For the second step (B, 256-640 ms), the first five traces are shown every 32 ms whereas the last two are shown each after 128 ms.
Results and Discussion Oxidation of Oxymyoglobin with Peroxynitrite Proceeds in Two Steps via Ferryl Myoglobin. The reaction of oxymyoglobin (MbFeO2) with peroxynitrite was studied by rapid-scan UV-vis spectroscopy between 510 and 660 nm at pH 6.7 and 20 °C. As shown in Figure 1, when a solution of MbFeO2 was reacted with an excess of peroxynitrite, the intensities of two absorption bands at 544 and 581 nm, characteristic of MbFeO2, rapidly decreased while a new band, characteristic of metmyoglobin (metMb), emerged with a maximum at about 630 nm. Two sets of isosbestic points were identified in the spectra at 526 and 589 nm (Figure 1A) and 519 and 612 nm (Figure 1B), respectively, which indicated the presence of an intermediate species during this transformation. As these wavelengths correspond to the isosbestic points between the spectra of MbFeO2 and ferryl myoglobin (MbFeIVdO) and those between the spectra of MbFeIVdO and metMb (32), respectively, ferryl myoglobin was assigned as the intermediate species. Further evidence for the formation of ferryl myoglobin (MbFeIVdO) was obtained by the identification, after addition of Na2S, of iron(II) sulfomyoglobin. This species has a strong absorbance band at 617 nm and is formed specifically from the reaction of MbFeIVdO with Na2S (33). Na2S (2 equiv) was first added to a MbFeO2 solution at pH 7.0, and as expected, no changes were observed in
Figure 2. UV-vis spectra of 100 µM MbFeO2 in 0.1 M phosphate buffer at pH 7 after the addition of Na2S and peroxynitrite: (1) MbFeO2 (100 µM), (2) MbFeO2 (100 µM), 200 µM Na2S, and 10 µM ONOO-, (3) MbFeO2 (100 µM), 200 µM Na2S, and 25 µM ONOO-, and (4) MbFeO2 (100 µM), 200 µM Na2S, and 100 µM ONOO-.
the UV-vis spectrum. In three separate experiments, 0.1, 0.25, or 1 equiv of peroxynitrite (relative to MbFeO2) was added to this solution. In all three cases, a new absorbance band with a maximum around 620 nm and an intensity proportional to the added amount of peroxynitrite could be observed (Figure 2). The occurrence of this characteristic band at a slightly higher wavelength than the expected 617 nm can be explained by the concurrent formation of some metmyoglobin (λmax ) 630 nm) (30).
ONOO--Mediated Oxidation of MbFeO2 and HbFeO2
Figure 3. Time courses measured at 612 and 589 nm for the two steps of the reaction of 2 µM MbFeO2 with 53 µM peroxynitrite in 0.05 M phosphate buffer at pH 6.7 and 20 °C. The traces correspond to the average of at least 50 single traces. The solid lines correspond to the best fits for the formation of the intermediate MbFeIVdO (612 nm) and for the formation of the final product metMb (589 nm). The resulting rate constants kobs are 4.1 ( 0.1 and 1.58 ( 0.03 s-1 (fit range of 1-5 s), respectively.
These data indicate that an excess of peroxynitrite is not a requirement for the formation of MbFeIVdO. The kinetics of the reaction of MbFeO2 with peroxynitrite were studied by single-wavelength stopped-flow spectroscopy under pseudo-first-order conditions in the pH range of 6.4-8.3 at 20 °C. Peroxynitrite was always present at least in a 10-fold excess to maintain pseudofirst-order conditions. This choice was dictated by the very small amplitudes obtained when MbFeO2 was used in excess, a consequence of the low yield of the reaction. Traces collected between the two isosbestic points, 589 and 612 nm, with either peroxynitrite or MbFeO2 in excess showed first an increase in the absorbance followed by a decrease. This observation further confirms the presence of an intermediate whose formation is independent of the reagent that is used in excess. The two steps of the reaction were studied separately. The first step (A), from MbFeO2 to MbFeIVdO, was studied by following the absorbance changes at 612 nm, the isosbestic point for the second step. The reaction of MbFeIVdO to metMb [second step (B)] was studied by following the absorbance changes at 589 nm, the isosbestic point for the first step. As shown in Figure 3, both time courses could be fitted to a single-exponential expression. The second traces were fitted only from the point when the first step was over, that is between 1 and 5 s for the example shown in Figure 3B. The observed rate constants (kobs) that were measured were both linearly dependent on the peroxynitrite concentration (Figure 4). The second-order rate constant values for the two steps decreased with increasing pH (Table 1). A third
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Figure 4. 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 at 20 °C and at different pH values. At pH 7.5, comparison with the data obtained from the reaction of separately prepared MbFeIVdO and peroxynitrite.
significantly slower transformation was observed when the measurements were carried out over a longer time scale (10-20 s). This reaction, probably due to oxidations of the globin by the large excess of peroxynitrite or by other reactive species formed in the preceding steps, was not investigated. To further support the assignment of the second step (B) of the peroxynitrite-mediated oxidation of MbFeO2 as the reduction of MbFeIVdO to metMb, we investigated the kinetics of the reaction of separately prepared MbFeIVdO with peroxynitrite at pH 7.5 by following the absorbance changes at 589 nm. As can be clearly seen from the example depicted in Figure 5, in contrast to the trace shown in Figure 3B, the time courses could be fitted well to a single-exponential expression from the beginning of the measurement. The data presented in Figure 4B and summarized in Table 1 show that MbFeIVdO reacts with peroxynitrite with a rate constant that is almost identical to that of the second step of the peroxynitrite-mediated oxidation of MbFeO2. Nitrite, always present in different amounts as a contaminant of the peroxynitrite solutions, is also likely to react with ferryl myoglobin. Stopped-flow kinetic studies of the reaction between nitrite and MbFeIVdO, carried out by following the absorbance changes at 589 nm, revealed that this reaction is significantly slower than the peroxynitrite-mediated reduction of MbFeIVdO. A value of 86 ( 6 M-1 s-1 was obtained for the second-order rate constant at pH 6.5 and 20 °C (Figure S1). These data, together with the observation that the oxidation of MbFeO2 to metMb by nitrite is even slower (34), indicate that the ubiquitous contaminant nitrite is not likely to have any influence on the kinetics
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Table 1. Summary of Second-Order Rate Constants Obtained at 20 °C HOONO
HOONO
pH
8 MbFeIVdO MbFeO2 9 k
8 MbFeIII MbFeIVdO 9 k
6.4 6.7 7.3 7.3a 7.3a,b 7.5 7.5b 8.3
(11.2 ( 0.3) × 104 M-1 s-1 (8.1 ( 0.2) × 104 M-1 s-1 (5.4 ( 0.2) × 104 M-1 s-1 (31.3 ( 0.9) × 104 M-1 s-1 (4.6 ( 0.2) × 104 M-1 s-1
(4.2 ( 0.1) × 104 M-1 s-1 (3.1 ( 0.1) × 104 M-1 s-1 (2.2 ( 0.1) × 104 M-1 s-1 (3.2 ( 0.1) × 104 M-1 s-1 (2.8 ( 0.3) × 104 M-1 s-1 (1.8 ( 0.1) × 104 M-1 s-1 (1.9 ( 0.1) × 104 M-1 s-1 (1.21 ( 0.04) × 104 M-1 s-1
1
HOONO
2
HOONO
pH
8 MbFeIVdO MbFeII 9 k
8 MbFeIII MbFeIVdO 9 k
7.5
∼106 M-1 s-1
(2.3 ( 0.1) × 104 M-1 s-1
1
2
NO2-
pH
8 MbFeIII MbFeIVdO 9 k
6.5
86 ( 6 M-1 s-1
2
HOONO
a
HOONO
pH
8 HbFeIVdO HbFeO2 9 k
8 HbFeIII HbFeIVdO 9 k
7.0
(8.8 ( 0.4) × 104 M-1 s-1
(9.4 ( 0.7) × 104 M-1 s-1
1
2
In the presence of 2.5 mM CO2. b Separately prepared MbFeIVdO.
Figure 5. Time course measured at 589 nm for the reaction of 2.2 µM MbFeIVdO with 25 µM peroxynitrite in 0.05 M phosphate buffer at pH 7.5 and 20 °C. The trace corresponds to the average of at least 50 single traces. The solid line corresponds to the best fit. The obtained rate constant kobs ) 0.54 ( 0.01 s-1.
and the mechanism of the peroxynitrite-mediated oxidation of MbFeO2. Iron(III)-porphyrin complexes have been reported to catalyze the isomerization of peroxynitrite to nitrate at pH 7.4 (35-37). The first step of this reaction involves the formation of a ferryl complex, the active species for the catalysis (35). Three possible structures have been proposed as intermediates for the isomerization reaction, two of which require two empty faces of the porphyrin. In our system, the reaction of ferryl myoglobin with peroxynitrite is not catalytic but yields the corresponding iron(III) form of the protein. The different reactivities toward peroxynitrite displayed by the iron-porphyrin complexes and Mb are very likely to be caused by the presence of the proximal imidazole bound to the heme. Another significant difference between the complexes and myoglobin is that, in contrast to other heme proteins (12, 20), the heme of Mb does not react with peroxynitrite in its iron(III) form (see also ref 25). True Reactants in the Oxidation of Oxymyoglobin with Peroxynitrite. As shown in Figure 4 and Table 1, the values of the second-order rate constants for both steps of the peroxynitrite-mediated oxidation of MbFeO2 decrease with increasing pH. A similar pH
dependence had already been observed for the overall peroxynitrite-mediated oxidation reaction of HbFeO2 to metHb (24). This pH dependence suggests that peroxynitrous acid is the species which reacts in the two steps of the reaction with MbFeO2 and MbFeIVdO, respectively. In addition, the observation that the Mb heme sits in a rather hydrophobic pocket (38) supports this hypothesis. Alternatively, the faster reaction rate observed at lower pH could be a consequence of the increased accessibility to the heme due to protonation of the distal histidine [pKa ≈ 6 (39)], which swings out toward the solvent and leaves the heme pocket in the “open conformation” (40). In analogy to the oxidation of oxymyoglobin with hydrogen peroxide (32), as the dissociation rate for dioxygen from MbFeO2 is relatively large [24.2 s-1 (41)], it is conceivable that the reaction of MbFeO2 with peroxynitrous acid proceeds through a two-electron oxidation of the dissociated deoxymyoglobin form (MbFeII) which is in equilibrium with MbFeO2 (Kd ) 1.09 × 10-6 M) (41) (eq 1). Rapid-scan UV-vis spectroscopic studies of the peroxynitrite-mediated oxidation of MbFeII were carried out between 350 and 450 nm at pH 7.5 and 20 °C. The results indicated that also this reaction proceeds in two steps. MbFeII is rapidly oxidized to MbFeIVdO, which reacts with peroxynitrite to form metMb. As seen in Figure 6, the maximum of the Soret band is shifted in about 30 ms from 434 (MbFeII) to 421 nm, the characteristic absorbance maximum for MbFeIVdO (trace 3). Reaction of MbFeIVdO with peroxynitrite yields metMb (trace 8, λmax ) 408 nm). Preliminary kinetic studies indicate that the value of the rate constant for the oxidation of MbFeII to MbFeIVdO at pH 7.5 and 20 °C is about 106 M-1 s-1. This value is consistent with that calculated by using a Kd of 1.09 × 10-6 M and an O2 concentration of 2.5 × 10-4 M and by considering MbFeII as the reactant in the peroxynitrite-mediated oxidation of MbFeO2. It has been reported that O2 dissociation rates drastically increase with decreasing pH, which is when the distal histidine is protonated and the “open conformation” of the heme pocket is observed (42). If O2 dissociation has to take place prior to reaction of MbFeO2 with peroxynitrous acid,
ONOO--Mediated Oxidation of MbFeO2 and HbFeO2
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Figure 6. Rapid-scan UV-vis spectra of the reaction of 8 µM MbFeII and 53 µM peroxynitrite in 0.05 M phosphate buffer at pH 7.5 and 20 °C. Traces are shown at the following measuring times: (2) 16 ms, (3) 32 ms, (4) 320 ms, (5) 640 ms, (6) 960 ms, (7) 1.28 s, and (8) 3.87 s.
this observation would represent a further explanation for the higher rate constants obtained at lower pH. The value of the rate constant measured for the second step, that is, the reduction of MbFeIVdO to metMb, is in agreement with those obtained when either MbFeO2 or MbFeIVdO is reacted with peroxynitrite (Table 1). These data thus indicate that, although we cannot exclude completely the possibility that MbFeO2 might react with peroxynitrous acid at a much slower rate, the oxidation of MbFeO2 by peroxynitrous acid is likely to take place according to eqs 1-3. Peroxynitrous acid may act as a two-electron oxidant toward MbFeII and generate MbFeIVd O and NO2- (eq 2). In the second step, ferryl myoglobin may oxidize HOONO to the peroxynitrite radical, ONOO• (eq 3). This is a thermodynamically feasible reaction as the estimated E° (ONOO•/ONOO-) is about 0.8 V (43), whereas E° (MbFeIVdO/metMb) is close to 1 V (31). Reactivity studies of MbFeIVdO (44) and oxoiron(IV)porphyrin complexes (45) suggest that the oxoiron(IV) group readily oxidizes substrates such as phenols via hydrogen atom abstraction. The reaction with HOONO may proceed in a similar way via the activated intermediate (MbFeIVdO‚‚‚H‚‚‚OONO). The fate of the peroxynitrite radical formed is unknown; in particular, it is not known whether it undergoes a reaction with the globin. A detailed analysis of the protein after the reaction might allow for the identification of possible modified amino acid residues. These studies are currently in progress in our laboratory. Recently, ab initio calculations have indicated that the ONOO• radical is unstable and is likely to dissociate to NO• and O2 according to eq 4 (46). However, by using a NO• analyzer with a chemiluminescent detector, we did not detect any nitrogen monoxide in the course of the peroxynitrite-mediated oxidation of MbFeO2. Kd
MbFeO2 {\} O2 + MbFeII
(1)
MbFeII + HOONO f MbFeIVdO + NO2-
(2)
MbFeIVdO + HOONO f metMb + ONOO•
(3)
ONOO• a NO• + O2
(4)
Influence of CO2. The rapid reaction between ONOOand CO2, which yields ONOOCO2- (13-15), represents
one of the most significant routes for peroxynitrite consumption in biological systems. We have studied the influence of an excess of CO2 on the peroxynitritemediated oxidation of MbFeO2. Preliminary results show that an excess of CO2 (1-5 mM) accelerates the oxidation of MbFeO2 to MbFeIVdO in a concentration-dependent way, whereas the second step of the reaction, that is, the reduction of MbFeIVdO to metMb, is only slightly accelerated by the presence of CO2 (Table 1). As the rate constant for the reaction of ONOO- with CO2 (15) is on the same order of magnitude as those for its reaction with oxymyoglobin and ferryl myoglobin, in the presence of a large excess of CO2, the adduct ONOOCO2- or one of its decomposition products are likely to be the species which react with the protein. The second-order rate constants for the two steps of the peroxynitrite-mediated oxidation of MbFeO2 in the presence of 2.5 mM CO2 at pH 7.3 are (31.3 ( 0.9) × 104 and (3.2 ( 0.1) × 104 M-1 s-1, respectively. As in the CO2free system, when separately prepared ferryl myoglobin was reacted with peroxynitrite in the presence of 2.5 mM CO2, an almost identical second-order rate constant was obtained for the second step of the reaction (Table 1). These results suggests that, even though the reaction rates are different, the same myoglobin species are formed with or without an excess of CO2. Ferryl myoglobin could be formed by a concerted two-electron oxidation of MbFeII to yield CO2 and NO2- (eq 5).
The mechanism for the second step, that is, the reduction of MbFeIVdO to metMb, is still unclear. More detailed studies, that is, analysis of the nitrogen-containing products and identification of possibly modified amino acid residues, are currently in progress in an effort to gain a better understanding of the mechanism of the overall reaction and, in particular, an explanation for the observation that the rate of the second step of the reaction is only slightly influenced by the presence of CO2. Oxidation of Oxyhemoglobin with Peroxynitrite. Analogous studies were carried out on the reaction of oxyhemoglobin (HbFeO2) with peroxynitrite. Rapid-scan UV-vis spectra, measured between 510 and 660 nm at pH 6.9 and 20 °C, are comparable to those obtained with oxymyoglobin (Figure S2). Two sets of isosbestic points were identified at 524 and 586 nm, and 522 and 609 nm, respectively, which suggested, again, the presence of a ferryl species, HbFeIVdO, as an intermediate in this system. Further evidence for the formation of ferryl hemoglobin (HbFeIVdO) was obtained from the identification, after addition of Na2S, of iron(II) sulfohemoglobin. As in the oxymyoglobin experiment, after addition of 1 equiv of peroxynitrite (relative to the heme concentration in HbFeO2) to a mixture of HbFeO2 and 2 equiv of Na2S, a new absorbance band with a maximum at 620 nm was observed (Figure S3). This absorbance is indicative of the formation of iron(II) sulfohemoglobin from the specific reaction between HbFeIVdO and Na2S. Single-wavelength stopped-flow spectroscopic studies were carried out under pseudo-first-order conditions with
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peroxynitrite in excess at pH 7.0 and 20 °C. The two steps of the reaction were followed at 586 and 609 nm, respectively. The observed rate constants that were measured were both linearly dependent on the peroxynitrite concentration (Figure S4). The second-order rate constant values obtained for the first and second steps were (8.8 ( 0.4) × 104 and (9.4 ( 0.7) × 104 M-1 s-1, respectively. The very small differences between the rate constants for the two steps of the reaction might explain why previously other groups did not identify the presence of the ferryl intermediate (24, 25). Indeed, these groups have studied this reaction by following the absorbance changes at 577 nm, the wavelength at which the difference in the extinction coefficients between oxyhemoglobin and methemoglobin is the largest. However, at this wavelength, the extinction coefficient of HbFeIVdO has a value between those of HbFeO2 and metHb. Time courses collected at this wavelength show a continuous decrease in absorbance and can be fitted, although not very well, to a single-exponential expression. Possible Biological Implications. Ferryl hemoglobin (or myoglobin) plus a transient protein radical, which has been proposed to be formed in vivo by reaction of methemoglobin (or myoglobin) with hydrogen peroxide, has been implicated in several types of oxidative damage (47, 48). The reaction of oxy- or deoxyhemoglobin (or myoglobin) with H2O2 yields the one-electron reduced ferryl species which, in particular for myoglobin, have also been shown to be able to oxidize a variety of substrates (47, 48). Ischemic conditions such as low oxygen tension, low pH, and high hydrogen peroxide concentrations (10 µM in ischemic heart muscle) (49) favor these oxidations. It has thus been proposed that ferryl myoglobin may be responsible for tissue injury resulting from reperfusion and reoxygenation of ischemic myocardium, and indeed, ferryl species have been detected in rat heart after a period of induced ischemia (50). As the rates of reaction of oxy- and deoxymyoglobin with H2O2 [kMbFeO2 ) 21 M-1 s-1 and kMbFeII ) 3.6 × 103 M-1 s-1 (32)] are at least 3 orders of magnitude smaller than the corresponding rates of reaction with peroxynitrite (Table 1), the reactions described in this paper may represent an alternative, not yet recognized, route for the formation of ferryl species in vivo. It has been proposed that peroxynitrite is formed when ischemic tissues are reperfused and that it is one of the species responsible for oxidative lesions found in these tissues (51). However, this work shows that MbFeII, which is also present in significant concentrations in these tissues, is among the species which react with the highest rate with peroxynitrite. This reaction, which is thus likely to take place in vivo, yields nitrite and another oxidizing species, MbFeIVdO. As we have also shown that MbFeIVdO is rapidly reduced by peroxynitrite, the overall reaction of deoxy- or oxymyoglobin with peroxynitrite may represent a detoxification pathway for this strong oxidant. Nevertheless, a better understanding of the mechanism of this reaction both in the absence and in the presence of an excess CO2 and, in particular, the identification of the products derived from the oxidation of peroxynitrite by ferryl myoglobin are needed to evaluate its possible relevance in vivo. These studies are currently in progress in our laboratory.
Exner and Herold
Acknowledgment. These studies were supported by the Swiss National Science Foundation and ETH Zu¨rich. We thank APEX Bioscience, Inc., for the supply of purified human hemoglobin. Supporting Information Available: Primary kinetic data for the nitrite-mediated reduction of MbFeIVdO (Figure S1) and for the peroxynitrite-mediated oxidation of HbFeO2 (Figures S2 and S4) and the UV-vis spectrum of sulfoHb (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.
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