bicarbonate and hydrogen peroxide

Free Radical Biology & Medicine, Vol. 35, No. 12, pp. 1538 –1550, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/j.freeradbiomed.2003.08.019

Original Contribution METHIONINE OXIDATION BY PEROXYMONOCARBONATE, A REACTIVE OXYGEN SPECIES FORMED FROM CO2/BICARBONATE AND HYDROGEN PEROXIDE DAVID E. RICHARDSON, CELESTE A. S. REGINO, HUIRONG YAO,

and JODIE

V. JOHNSON

Center for Catalysis, Department of Chemistry, University of Florida, Gainesville, FL, USA (Received 1 April 2003; Revised 31 July 2003; Accepted 28 August 2003)

Abstract—Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO4⫺, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO2. The kinetic parameters for HCO4⫺ oxidation of free methionine have been obtained (k1 ⫽ 0.48 ⫾ 0.08 M⫺1s⫺1 by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood (⬃25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H2O2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of ␣1-proteinase inhibitor (␣1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO4⫺ oxidation of ␣1-PI (0.36 ⫾ 0.06 M⫺1s⫺1) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the ␣1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized ␣1-PI. These results strongly suggest that HCO4⫺ should be considered a reactive oxygen species in aerobic metabolism. © 2003 Elsevier Inc. Keywords—Reactive oxygen species, ROS, Methionine, Methionine sulfoxide, Hydrogen peroxide, Peroxymonocarbonate, ␣1-proteinase inhibitor, ␣1-PI, ␣1-antitrypsin, Free radicals

INTRODUCTION

mulation of metSO residues in the tissue has also been associated with aging [6]. For example, the human juvenile lens protein contains little metSO, but the metSO content rises with age [7]. In cataractous lenses, about 45% of the total met residues and 60% total cysteine residues are oxidized [7–9]. In another example, studies of the calcium-binding protein calmodulin showed that approximately six out of nine met residues are oxidized to metSO in the brains of aged Fischer rats, but calmodulin met residues in the brains of young rats are essentially unoxidized [10]. This met oxidation corresponded to the age-dependent reduction of the ability of calmodulin to activate plasma membrane Ca-ATPase [10,11], and the activity can be restored by treatment of the oxidized calmodulin with metSO reductase [12]. Our recent kinetic investigations of bicarbonate-catalyzed organic sulfide oxidations [13] strongly support the identification of peroxymonocarbonate (HCO4⫺) as the direct oxidant. Peroxymonocarbonate is formed from

In this report, we provide kinetic evidence that peroxymonocarbonate ion, HCO4⫺, is a previously unrecognized reactive oxygen species (ROS) in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate, both of which are ubiquitous in aerobic biochemistry. We have focused initially on the possible role of peroxymonocarbonate in the biological oxidation of the highly reactive sulfur nucleophile of methionine (met) in proteins. The presence of methionine sulfoxide (metSO) in proteins is an established marker of oxidative stress [1–3], which is associated with the degenerative effects of many diseases, including AIDS [4,5]. AccuAddress correspondence to: David E. Richardson, Department of Chemistry, University of Florida, 212 Leigh Hall, Gainesville, FL 32611-7200, USA; Tel: (352) 392-6736; Fax: (352) 392-3255; E-Mail: [email protected]. 1538

Methionine oxidation by peroxymonocarbonate

1539

Fig. 1. Scheme for the activation of H2O2 by HCO3⫺/CO2 in the oxidation of methionine. Hydration (right side) and the analogous perhydration (left side) pathways are shown. At physiological pH values, the pathway involving direct reaction of HO2⫺ with CO2 is likely to be significant (center). The hydration/dehydration of CO2 is catalyzed by a functional zinc carbonic anhydrase model compound, thereby increasing the rate of substrate oxidation when k1 is large (see text).

bicarbonate and hydrogen peroxide in the absence of added catalysts and subsequently oxidizes the substrate (Eqns. 1 and 2).

H2O2 ⫹ HCO3⫺ º H2O ⫹ HCO4⫺ Keq ⫽

⫺ 4

[HCO ] [H2O2] [HCO3⫺]

RSR⬘ ⫹ HCO

⫺ 3

3 RS(O)R⬘ ⫹ HCO k1

d[RSR⬘] k1 kf [HCO3⫺]0 [H2O2]0 [RSR⬘] ⫽ dt kr ⫹ kl 关RSR⬘兴 ⫹ kf 关H2O2兴0

(3)

If the equilibrium reaction of Eqn. 1 is rapid compared to the oxidation of Eqn. 2 (i.e., kr ⫹ kf[H2O2]0 ⬎⬎ k1[RSR⬘]) then Eqn. 3 reduces to a pre-equilibrium rate law (Eqn. 4).

⫺ 4

⫽

⫺

kf (1) kr

⫺

d[RSR⬘] k1 Keq [HCO3⫺]0 [H2O2]0 [RSR⬘] ⫽ dt 1 ⫹ Keq [H2O2]0

(4)

(2)

Thus far, all kinetic results [13,14] are consistent with the equilibration reaction of Eqn. 1 (Keq ⫽ 0.32 in water at 25°C) proceeding via the perhydration of CO2 formed in equilibrium with bicarbonate (Fig. 1). Treating HCO4⫺ as a steady state intermediate in the mechanism of Eqns. 1 and 2 leads to the rate law of Eqn. 3, where H2O2 is in large excess and the bicarbonate concentration is expressed as an initial concentration, [HCO3⫺]0.

In our analysis of the kinetics of aryl sulfide oxidations by HCO4⫺ [13], we used a pre-equilibrium model modified to account for the formation of carbonate esters when primary alcohols are used as co-solvents. Given the facile nature of Eqn. 1, it is reasonable to expect that HCO4⫺ could be found throughout biology. The reactivity of peroxymonocarbonate toward sulfides at moderate temperature and its stability near neutral pH suggested a possible significance as an ROS. To our knowledge, peroxymonocarbonate has only been men-

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D. E. RICHARDSON et al.

tioned briefly as an ROS [15], and no supporting data were provided. In our investigation of HCO4⫺ biochemistry reported here, we have investigated the rates and mechanism of the in vitro bicarbonate-catalyzed hydrogen peroxide oxidation of met, both free and in ␣1-proteinase inhibitor (␣1-PI, formerly known as ␣1-antitrypsin). Further evidence for met oxidation, specifically involving Met358 and Met351 of the ␣1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized ␣1-PI. The oxidation of met in ␣1-PI, particularly Met358 [16], results in the decrease in the inhibitory activity of ␣1-PI toward a wide spectrum of serine proteases [17,18]. Unchecked protease activity in tissues can lead to various pathogeneses including adult respiratory distress syndrome [19,20], chronic obstructive pulmonary disease [17,21,22], emphysema [21–26], and rheumatoid arthritis [1,17,18,22]. MATERIALS AND METHODS DL-Methionine

was obtained from Eastman Chemical Company (Kingsport, TN, USA) and was used as received. Hydrogen peroxide (70% from Solvay Interox, Houston, TX, USA; 35% from Aldrich, St. Louis, MO, USA) was standardized iodometrically. Trihydroxylmethylamine (Tris), dithiothreitol (DTT), ammonium bicarbonate, and ammonium phosphate (both dibasic and monobasic forms), all analytical grade were obtained from Sigma (St. Louis, MO, USA) and was used as received. The macrocycle 1,4,7,10-tetraazacyclododecane ([12]aneN4, cyclen) was obtained from Aldrich and were used as received. Sodium hydroxide, sodium phosphate (monobasic, dibasic, and tribasic forms), sodium chloride, sodium bicarbonate, sodium carbonate, ammonium sulfate, phosphoric acid, and hydrochloric acid (36% by weight) were all analytical grade and were obtained from Fisher (Atlanta, GA, USA); they were used as received. Formic acid (70%) sequence grade was obtained from Fisher. Sepharose 4B, diethylaminoethyl cellulose (fibrous form) anion exchange resin, and blue dextran were obtained from Sigma. Water was purified by using a Barnstead E-Pure 3-Module Deionization System (water resistance ⱖ17 megohm-cm). Porcine pancreatic elastase (EC 3.4.21.36) type IV, endoproteinase Lys-C (EC 3.4.21.50) from Lysobacter enzymogenes, and bovine liver catalase (EC 1.11.1.6) were obtained from Sigma and were used as received. Human ␣1-PI (Sigma) was purified according to the procedures by Travis and coworkers [27,28]. Our procedure deviates from the literature procedure in that an isocratic elution rather than gradient elution is used for the Sepharose-blue dextran column step (1.0 ⫻ 30 cm

column, 200 ml of 2.0 mM NaCl in 50 mM Tris-HCl buffer pH 8.00 at 4°C with a flow rate of 1 ml/min). Purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The synthetic elastase substrate N-succinyl-L-trialanyl-p-nitroanilide [Suc-(Ala)3-Nan] was obtained from Sigma and was used as received. Proton and 13C nuclear magnetic resonance (NMR) spectra were obtained on a Gemini 300 MHz spectrometer at 25 ⫾ 0.1°C. Deuterated water (99.9%) and 13Cenriched sodium bicarbonate (99%) used for the NMR experiments were from Cambridge Isotope Laboratory, Inc. (Andover, MA, USA). Ultraviolet (UV)-visible spectrophotometric measurements were obtained on a Hewlett-Packard 8453 spectrophotometer. Temperature was maintained at 25 ⫾ 0.1°C by using a Fisher Scientific Isotemp 1600S water bath circulator. Amino acid analyses were provided by the Protein Chemistry Core Facility, ICBR of the University of Florida. Oxidation of free met The oxidation of met (10 mM) with hydrogen peroxide (0.10 M) in D2O was followed by 1H NMR. A stock solution of the 1,4,7,10-tetraazacyclododecanezinc(II), [([12]aneN4)ZnII(OH2)](ClO4)2, was prepared in situ by dissolving [12]aneN4 (8.6 mg, 0.050 mmol) and Zn(ClO4)2 · 6 H2O (18.6 mg, 0.050 mmol) in 2.0 ml of deuterated water. The mixture was stirred for 1–2 h before each measurement. Solutions of met (10 mM) and ammonium bicarbonate (10 mM) were mixed with or without the stock solution of the zinc(II) complex (5.0 mM) immediately before each measurement. The oxidation was followed by the decreasing intensities of the methyl protons of the substrate met. Similar experiments were performed for the background reactions, wherein a buffer solution of ammonium hydrogen phosphate (I ⫽ 0.10) replaced the solution of ammonium bicarbonate in the above reactions. In addition, met oxidation with a pre-equilibrated solution of hydrogen peroxide in bicarbonate buffer was investigated. The rate of oxidation is not different from that of the standard procedure. It was also confirmed that removal of trace metal contaminants (by addition of diethylenetriamine pentaacetic acid or by chelate resin) had no effect on the reaction rates. Oxidation of met was also followed by monitoring the decrease in the absorbance of hydrogen peroxide at 275 nm over time. The dependence of the rate of met oxidation on [HCO3⫺] and on [H2O2] was investigated. Oxidation of ␣1-proteinase inhibitor An experimental approach based on published procedures [29,30] was used. Stock solutions of hydrogen peroxide, with (NH4)2HPO4 added for the background

Methionine oxidation by peroxymonocarbonate

reactions or with NH4HCO3 added for the catalyzed reactions, in 0.20 M Tris-HCl at pH 8.0 were pre-equilibrated for 1 h. Quantitative amounts of the oxidant solutions were then added to a solution of ␣1-PI in 0.20 M Tris-HCl buffer at pH 8.0. Final conditions for the oxidation reactions were [␣1-PI] ⬇ 13 ␮M, [H2O2] ⫽ 10 mM, [(NH4)2HPO4] ⫽ 0.10 M or [NH4HCO3] ⫽ 0.10 to 0.70 M, 0.20 M Tris-HCl (pH 8.0) at 25°C. Aliquots of the reaction mixture were then taken at different time intervals and added to a solution of catalase (2 nM) in 0.20 M Tris-HCl buffer (pH 8.0) to quench the reaction. Elastase in 0.20 M Tris-HCl (pH 8.0) was added to the quenched reaction and equilibrated for 2 h to allow the elastase-inhibitor complex to form. The inhibitory activity of ␣1-PI on elastase was monitored by measuring the proteolytic activity of elastase on Suc-(Ala)3-Nan (20 mM stock solution in dimethylformamide). Final conditions for the elastase inhibitory assay were: [␣1-PI]total ⫽ 0.03 ␮M, [elastase] ⫽ 0.10 ␮M and [substrate] ⫽ 0.33 mM, all in 0.20 M Tris-HCl buffer (pH 8.0) at 25°C. The release of the hydrolysis product p-nitroaniline was monitored by measuring the increase in absorbance at 410 nm. The decomposition of HCO4⫺ by catalase was observed by using 13C NMR of a pre-equilibrated solution of 13C-enriched bicarbonate (1.0 M) with H2O2 (0.10 M). The peak for HCO4⫺ disappeared upon addition of the catalase solution (1.0 nM) in less than 1 min [31]. This experiment confirmed that the oxidation reaction is quenched upon the addition of the catalase as both the HCO4⫺ and H2O2 are decomposed. Separate 1H NMR experiment confirmed that the Tris buffer is not oxidized under the conditions of these experiments. SDS-PAGE was used to confirm the formation of the ␣1-PI-elastase complex. Mixtures of purified ␣1-PI (0.15 ␮M) and elastase (0.10 ␮M) equilibrated for at least 2 h were run in 12% T/2.7% C with 0.10% SDS gel electrophoresis. The gel was stained with Coomassie brilliant blue G. Similar experiments were performed using the oxidized ␣1-PI instead of the native ␣1-PI. CNBr cleavage and MetSO analysis Simultaneous analysis of met and metSO was based on previously established procedures [32,33]. A cyanogen bromide (CNBr) solution was freshly prepared as a 10 M stock in acetonitrile and diluted to 100 mM by using 70% formic acid. Briefly, 100 ␮l of 100 mM CNBr was added to an oxidized sample of purified ␣1-PI. The glass vial was then wrapped in aluminum foil and incubated overnight at room temperature in a fume hood. After 18 h, the solution was lyophilized and then hydrolyzed in 6 M HCl at 110°C for 24 h in the presence of 1.5 mM DTT. The samples were then used for amino acid

1541

analysis by using a precolumn derivatization phenylisothiocyanate method on an Applied Biosystem Model 420A instrument. Peptide mapping of the oxidized ␣1-PI using HPLC/UV/ESI/QIT-MS An experimental approach based on published procedures by Levine and coworkers [33] was used. Briefly, 25 ␮l of 0.20 M Tris-HCl with 2.0 mM ethylenediaminetetraacetic acid buffer at pH 8.5 was added to the lyophilized oxidized sample of purified protein followed by 25 ␮l of hexafluoroisopropanol as a denaturant. The solution was then lyophilized for 6 h, and 50 ␮l of water was then added. Endoproteinase Lys-C (20 ␮l) was then added to the lyophilized sample, which was then incubated at 37°C overnight. The digest was then cooled and analyzed via high-performance liquid chromatography (HPLC)/UV/electrospray ionization (ESI)/quadrupole ion trap-mass spectrometry (QIT-MS) with both spectrophotometric (Applied Biosystems Model 785A Programmable Absorbance Detector at ␭ ⫽ 220 nm) and mass spectrometric detection. The MS data were acquired with a ThermoFinnigan MAT LCQ (San Jose, CA, USA) in ESI mode. Typical operating parameters were an ion spray voltage of 3.5 kV, capillary voltage of 15 V, and capillary temperature of 240°C. MS data were acquired in the m/z range of 400 –2000. The mobile phase was provided by a Beckman Instruments (Fullerton, CA, USA) System Gold model 126 pump. A binary system with mobile phase A as 0.5% acetic acid in water and B as 0.5% acetic acid in methanol was used. A Waters (Milford, MA, USA) Symmetry Shield RP18 (2.1 ⫻ 150 mm ⫻ 3.5 ␮m) column with no guard column was used for chromatographic separation at a flow rate of 0.2 ml/min. RESULTS

Free met oxidation The rates of bicarbonate-catalyzed oxidations of met were investigated by using 1H NMR (Fig. 2). With met concentrations suitable for accurate NMR analysis (ⱖ10 mM), the effective rate constant for the reaction in Eqn. 2 (i.e., k1[RSR⬘]) is comparable to the rate constant kr for the reverse of Eqn. 1, so the pre-equilibrium kinetic model we used previously [13] cannot be applied. Instead, numerical simulations and nonlinear regression were used to fit the kinetic data. In the NMR experiments, both H2O2 and HCO3⫺ were 0.10 – 0.20 M. The determined second-order rate constants are: k0 ⫽ (6 ⫾ 1) ⫻ 10⫺3M⫺1s⫺1 (direct oxidation of met by H2O2) and k1 ⫽ 1.0 ⫾ 0.3 M⫺1s⫺1 (Eqn. 2). Using spectrophotometric measurements to obtain the initial rate, the values ob-

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D. E. RICHARDSON et al.

Fig. 2. Time dependence of methionine oxidation as observed by 1H nuclear magnetic resonance (NMR) in D2O. In all reactions, [H2O2] ⫽ 0.10 M, [RSMe] ⫽ 0.010 M. (●) Background or uncatalyzed reaction: (NH4)2HPO4, ␮ ⫽ 0.10, pH ⫽ 8.0; (}) Bicarbonate-activated oxidation: [NH4HCO3] ⫽ 0.10 M, pH 8.0. (Œ) Zinc-catalyzed (see text): [ZnL] ⫽ 5.0 mM (5% of [H2O2]), [NH4HCO3] ⫽ 0.10 M, pH 8.0. Lines are fits from numerical kinetic simulations (see text). Data points are from a single NMR experiment for each reaction condition. Error for each point is ⬃5% based on multiple experiments.

tained for the second-order rate constants are: k0 ⫽ (7.5 ⫾ 0.7) ⫻ 10⫺3M⫺1s⫺1 and k1 ⫽ 0.48 ⫾ 0.08 M⫺1s⫺1. These latter results are comparable to the ones obtained from the 1H NMR experiments. At the high levels of met used, catalysis of Eqn. 1 was apparent from the numerical simulations of the experimental data, presumably due to nucleophilic catalysis by met. In the absence of substantial dehydration catalysis by met, the bicarbonate-activated oxidation would be similar to the uncatalyzed reaction as kf in Eqn. 1 would become rate-limiting based on the rate constants for Eqn. 1 derived in the absence of substrate [13]. The catalysis of Eqn. 1 by 1,4,7,10-tetraazacyclododecanezinc (II) was investigated with regard to the effect on the met oxidation (Fig. 2). Some acceleration of the oxidation is evident, and the experimental data are fit well by a pre-equilibrium rate law (Eqn. 4). The derived value of k1 (0.9 M⫺1s⫺1) is the same as that obtained via numerical simulation of the data in the absence of the zinc complex. The acceleration of the oxidation by 5 mM ZnL is consistent with its catalysis of Eqn. 1 [14], but the increase is not large because met also catalyzes the dehydration of bicarbonate. Oxidation of ␣1-proteinase inhibitor The residual elastase activity under our assay conditions depends on the prior exposure time of the proteinase inhibitor to H2O2 and the concentration of HCO3⫺ in the oxidizing medium (Fig. 3). With increasing exposure to H2O2, the inhibitory activity of ␣1-PI is reduced, in agreement with the literature [16,29,34,35]. The rate of inhibition loss increased with increasing bicarbonate in

the oxidation step. At maximum oxidation reaction time and the highest concentrations of HCO3⫺, the residual elastase activity approached that of the native enzyme in the absence of the inhibitor, indicating that ␣1-PI inhibition was essentially eliminated by complete oxidation. SDS-PAGE of the oxidized ␣1-PI and elastase mixture (data not shown) is consistent with previous work by Travis and coworkers [16], and it shows the elimination of the characteristic ␣1-PI/elastase adduct following complete oxidation of ␣1-PI. We developed a kinetic model for the data to fit the observed rates and thereby allow comparison of the kinetic parameters for ␣1-PI oxidation to those above for free met oxidation. Kinetic model for inhibitor assay The relevant reactions used to model the results for the oxidation of ␣1-PI are given in Eqns. 5– 8 where E ⫽ elastase, S ⫽ Suc-(Ala)3-Nan substrate, P ⫽ p-nitroaniline product, ␣1-PI ⫽ active form of the inhibitor, and ␣1-PIox ⫽ oxidized form of the inhibitor. E⫹S 3 P

kE ⫽ 共kcat/KM兲 关S兴 (5)

␣1-PI ⫹ E 3 关E-␣1-PI]

irreversible

(6)

␣1-PI ⫹ H2O2 3 ␣1-PIox ⫹ H2O

k0

(7)

␣1-PI ⫹ HCO4⫺ 3 ␣1-PIox ⫹ HCO3⫺

k1

(8)

The rate constant for appearance of product is given by the first-order constant kE because [S] ⬍⬍ KM under our

Methionine oxidation by peroxymonocarbonate

1543

Fig. 3. Plots based on Eqn. 11 summarizing loss of inhibitory activity of ␣1-PI on elastase in the course of oxidation by H2O2 with increasing amounts of bicarbonate and total time of exposure to the oxidant solution. At long exposure times and high bicarbonate concentration, the inhibition by ␣1-PI is negligible and the maximum rate of substrate hydrolysis (kE[E] ⫽ 1.4 s⫺1[E]) is approached. Oxidation reaction conditions are: [␣1-PI] ⬇13 ␮M, [H2O2] ⫽ 10 mM, [HCO3⫺] ⫽ 0 (■), 0.10 M (⽧), 0.30 M (Œ), 0.50 M (‚), and 0.70 M (䊐). The model curves are based on a nonlinear regression fit described in the text (for [HCO3⫺] ⫽ 0, 0.10, 0.30, 0.50, 0.70 M from lower curve to top curve). Only single data points were obtained at t ⫽ 1200 s. Assay conditions are: [␣1-PI] ⫽ 0.03 ␮M, [E]0 ⫽ 0.10 ␮M, [S] ⫽ 0.33 mM in 0.20 M Tris-HCl buffer at pH 8.0.

conditions (KM is the Michaelis constants in M units) [36]. The binding constant of ␣1-PI to elastase is exceptionally large [34,36], and Eqn. 6 can be treated as an irreversible reaction under our conditions (oxidized ␣1-PI does not significantly inhibit porcine pancreatic elastase). The concentration of native ␣1-PI after incubation time t is given by [␣1-PI]t ⫽ [␣1-PI]0 exp 共⫺kobs t兲

(9)

where kobs is expressed as Eqn. 10, which can be obtain by adding the background rate (k0[H2O2][RSR⬘]) to Eqn. 4. kobs ⫽

k1 Keq 关HCO3⫺兴0 [H2O2]0 ⫹ k0 [H2O2]0 1 ⫹ Keq [H2O2]0

(10)

The second term in Eqn. 10 is the direct oxidation of ␣1-PI by H2O2, and the first term is based on our previous kinetic model for sulfide oxidation by bicarbonate-

activated H2O2 [13] (a pre-equilibrium model can be used here because k1[S] ⬍⬍ kr). The first term in Eqn. 10 reduces to k1Keq[HCO3⫺][H2O2] when peroxide concentration is low, as in our conditions. The observed rate of hydrolysis is then given by Eqn. 11, where [E]0 is the initial elastase concentration.

␯ ⫽ d 关P兴/dt ⫽ kE 兵关E兴0 ⫺ [␣1-PI]0 exp 共⫺kobs t兲其

(11)

Note that in our assay [␣1-PI]0 ⬍ [E]0, so at t ⫽ 0 (unoxidized ␣1-PI) elastase activity is observed. The data are fit well by the model (Fig. 3), and the values obtained by nonlinear regression analysis of the data with Eqn. 11 are k0 ⫽ 0.014 ⫾ 0.003 M⫺1s⫺1 and k1 ⫽ 0.36 ⫾ 0.06 M⫺1s⫺1. The fraction of the oxidation carried by HCO4⫺ under conditions of low peroxide concentration is given by the model as k1Keq[HCO3⫺]/ (k1Keq[HCO3⫺] ⫹ k0). At the highest concentration of bicarbonate, 0.7 M, ca. 85% of the oxidation is by peroxymonocarbonate.

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D. E. RICHARDSON et al. Table 1. Comparative Amino Acid Composition of Native and Oxidized ␣1-Proteinase Inhibitor (␣1-PI) Oxidized ␣1-PI

Residuea Glycine Homoserine Histidine Arginine Threonine Methionine Phenylalanine

Theoreticalb

Nativec

Uncatalyzedc

Catalyzedc

22 9 13 7 30 0 27

24.9 ⫾ 2.7 11.5 ⫾ 3.7 11.7 ⫾ 2.5 7.6 ⫾ 3.3 28.4 ⫾ 2.9 0 29.3 ⫾ 2.4

19.9 ⫾ 1.4 6.5 ⫾ 3.6 7.5 ⫾ 0.8 7.2 ⫾ 0.9 32.2 ⫾ 2.2 0.9 ⫾ 0.5 28.6 ⫾ 1.8

21.9 ⫾ 2.3 4.7 ⫾ 3.0 7.8 ⫾ 4.1 7.6 ⫾ 4.3 32.3 ⫾ 1.6 4.2 ⫾ 0.7 27.1 ⫾ 3.0

a Selected residues shown from the full analysis. Other amino acids were not significantly altered between native and oxidized ␣1-PI. Homoserine and methionine contents are changed significantly upon oxidation. b Composition from the primary sequence of ␣1-PI. c Average of the analyses after cyanogen bromide and hydrochloric acid hydrolyses in the presence of 1.5 mM dithiothreitol. Errors are reported as standard deviations.

A linear form of the rate equation can be derived for the oxidation assay. After rearranging, the logarithmic form of the rate Eqn. 11 leads to Eqn. 12. ln 共kE 关E兴0 ⫺ ␯兲 ⫽ ln 共kE [␣1-PI]0兲 ⫺ kobs t

(12)

Equation 12 shows that a plot of ln(kE[E]0⫺ ␯) vs. t will be linear with slope ⫺kobs (the equation is undefined at infinite reaction time). CNBr cleavage and metSO analysis The presence of metSO in oxidized ␣1-PI was demonstrated by the simultaneous analysis for met and metSO by amino acid analysis of the purified native and oxidized protein. Oxidation of other amino acids by hydrogen peroxide or by peroxymonocarbonate was also checked through an amino acid analysis. In the CNBr step, unoxidized mets were converted to homoserines while metSOs were left intact. DTT was added in the acid hydrolysis step to reduce the metSOs back to mets. The amount of met in the amino acid analysis therefore corresponds to the amount of metSO residues in the oxidized inhibitor, and the amount of homoserine in the amino acid analysis corresponds to the amount of unoxidized met residues. Results of the amino acid analysis (Table 1) indicated that an average of ⬃1 out of 9 mets were oxidized in the uncatalyzed oxidation reaction of ␣1-PI, as determined from the met analysis. This value is consistent with the previous studies on the oxidation of the solvent-exposed mets, Met351 and Met358, of the reactive center loop [16,33]. In the bicarbonate-catalyzed oxidation of ␣1-PI, ⬃4 mets were oxidized on average. Trends in the homoserine analyses (i.e., derived from unoxidized mets) were consistent with the met analyses, but the errors in the values are much higher.

Peptide mapping of the oxidized ␣1-PI In order to demonstrate the oxidation of the Met358 in the reactive center loop, a MS analysis of the peptide digest of the protein was performed. Endoproteinase Lys-C (Endo Lys-C) was used to cleave the protein at the C-side of lysines [37]. The primary sequence of ␣1-PI containing 394 residues with 34 lysine residues is shown in Fig. 4. Thirty-four fragments would be expected from a perfect digestion by Endo Lys-C; however, many more fragments could be expected from incomplete digestion. The possible combinations of the fragments at different charge states that could be observed in ESI-MS including [M ⫹ nH]n⫹ ions as well as other possible cationic (e.g., Na⫹) adducts were generated using the program FragESI [38] to aid in the MS peak identification. Approximately 70% of the total ion charge-normalized intensities in the native and oxidized proteins were assigned by using the FragESI program. Using the FragESI program, a peak observed at m/z ⫽ 1130 is assigned to the doubly charged (⫹2) unoxidized native fragment 30 (Fig. 5). A shift in m/z value of ␦m/z ⫽ 8 is predicted for the doubly charged ion for each oxygen being added to the fragment [i.e., conversion of met to metSO, with m/z ⫽ 1138 for the mono-oxidized and m/z ⫽ 1146 for the dioxidized fragment 30 (Fig. 5)]. Quantification of the peptides containing fragment 30 (and other oxidized fragments) was accomplished by integrating the total area of the mass chromatograms of the specific m/z ion and all of its observed charge states (i.e., ⫹1, ⫹2, ⫹3). Analysis of the oxidized ␣1-PI digests indicated that the mets (Met 351 and Met 358) in the reactive center loop (ions containing fragment 30) were either singly or doubly oxidized (Fig. 6), consistent with the loss of the inhibitory activity of ␣1-PI toward elastase [16,18,35]. This result was expected, as both mets are solvent ex-

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Fig. 4. Primary sequence of ␣1-PI. Lysine (K) residues are in bold and underlined. Thirty-four fragments are produced by cleavage at all lysine residues. Methionine (M) residues are underlined, and the methionines in the reactive center loop are in bold and underlined.

posed [39,40]. The native protein also showed some limited oxidation after 8 h exposure to air, indicating that the reactive center is easily oxidized, consistent with the decrease of inhibitory activity of the protein upon long exposure to air at room temperature. Another met that is partially exposed and that can presumably be oxidized is Met226 [18]. The MS analysis of the proteolytic digest showed oxidation in the fragment containing Met226 (fragment 18), further supporting the amino acid analysis indicating that a third met was oxidized in the catalyzed reaction. Other mets in the protein are well buried and unexposed, and they were not significantly oxidized according to the MS data. DISCUSSION

Peroxymonocarbonate as a heterolytic oxidant Peroxymonocarbonate is moderately reactive heterolytic oxidant that can be classified as an anionic peracid. Thermodynamic results for Eqn. 1 (Keq ⫽ 0.3 in water at 25°C) give a value of E° (HCO4⫺/HCO3⫺) ⫽ 1.8 ⫾ 0.1

V (vs. NHE), and HCO4⫺ is, therefore, a potent oxidant in aqueous solution [13]. The peroxymonocarbonate ion has been isolated in various salts and characterized by vibrational spectroscopy [41,42] and radiograph crystallography (KHCO4 · H2O2) [43]. The structure of the ion is that of a true peroxide, i.e., HOOCO2⫺, and it can be considered to be the CO2 adduct of OOH⫺ (Fig. 1). Oxidation of met In the oxidation of organic substrates such as sulfides, the acceleration of HCO4⫺ equilibration with H2O2 and HCO3⫺ can increase the rate of oxidation for more nucleophilic substrates (i.e., those with large k1 values for Eqn. 2) because the forward reaction of Eqn. 1 can become rate-limiting when k1[S] ⬎⬎ kr ⫹ kf[H2O2] (Eqn. 3). For most aryl sulfides, the pre-equilibrium model Eqn. 4 is appropriate. Dialkyl sulfides, such as mets, are stronger nucleophiles, and catalysis of Eqn. 1 can be an effective method for increasing the oxidation rate. The second-order rate constant k1 for oxidation of met is 100-fold greater for HCO4⫺ relative to the rate con-

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Fig. 5. High-performance liquid chromatography/electrospray ionization/mass spectrometry of the endopeptidase Lys-C digest of ␣1-PI showing the oxidation of the reactive center loop (fragment 30). Top spectrum shows the native doubly (m/z ⫹ 1130) and triply charged (m/z ⫽ 754) unoxidized fragment 30. Bottom spectrum shows the doubly (m/z ⫽ 1146) and triply charged (m/z ⫽ 765) dioxidized fragment 30 in which both methionines in the fragment have been oxidized.

stant k0 for oxidation by H2O2. The second-order rate constants for various aryl sulfide oxidations by HCO4⫺ are ⬃300-fold greater than those for H2O2 [13], and these increases in reactivity over that of H2O2 are quan-

titatively consistent with expectations based on a Brønsted analysis of the kinetics for other heterolytic peroxide oxidations. We have proposed [13,44] the detailed solvent-assisted SN2 mechanism of Scheme 1 for oxidation

Methionine oxidation by peroxymonocarbonate

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sulfides by bicarbonate-activated peroxide (data not shown). This result can be rationalized noting that the strong nucleophilic nature of sulfides leads to rapid oxidation by electrophilic heterolytic oxidants, such as peroxymonocarbonate and other peracids, and metals that tend to promote radical production (e.g., Fenton chemistry) are not required for rapid sulfoxide formation. Oxidation of ␣1-proteinase inhibitor

Fig. 6. Relative abundance of the oxidized fragments in oxidized ␣1-PI. See Fig. 4 for sequence and fragment identification. Fragment 28 –31 is the mono-oxidized fragment and fragment 30 is the dioxidized fragment, both of which contain the reactive center loop with Met358 (and Met 351). Fragment 18 –19 is the mono-oxidized fragment containing the partially accessible Met226. The total mass area is calculated from the sum of the mass chromatogram area of the specific m/z ion and all the charge states including those generated from [M ⫹ nNa⫹]. Sample ID 1 corresponds to the native ␣1-PI control protein with no oxidant, 2 to the uncatalyzed H2O2 oxidation of ␣1-PI, and 3 to the bicarbonatecatalyzed oxidation of ␣1-PI by H2O2. Results shown are for a single digest of each sample type.

of sulfides in water based on the solvent dependence of the oxidation kinetics, the solvent kinetic isotope effect, and analogies to other heterolytic peroxide oxidations. In this mechanism, water accelerates the oxygen transfer to the nucleophilic sulfide by assisting in the displacement of the carbonate leaving group. As expected from the mechanism, the kinetics are consistent with acceleration of Eqn. 1 by a zinc catalyst that mimics the reactivity of carbonic anhydrase. The net effect of the added zinc catalyst is not as large as that expected due to the met catalysis of Eqn. 1. However, in the presence of the catalyst, the equilibration reaction of Eqn. 1 becomes sufficiently rapid to be treated as a pre-equilibrium. Addition of Fe(2⫹) and Mn(2⫹) salts up to 10 ␮M had no significant effect on the oxidation of

As a model for a possible role for HCO4⫺ in vivo, the in vitro oxidation of the human neutrophil elastase inhibitor ␣1-PI [17] by H2O2 in the presence of bicarbonate ion was investigated. The oxidation was assayed by the inhibition of elastase-catalyzed hydrolysis of Suc(Ala)3-Nan, which has been studied previously [29,30]. It is known that the oxidation of ␣1-PI leads to a significant reduction in its inhibition of elastase [16,18,45], and this oxidation has been suggested to be an important regulatory mechanism in the activation of elastin breakdown by neutrophil elastase in the lung [25]. Reduced inhibitory activity has been associated with the oxidation of Met358 in the reactive center loop of ␣1-PI [16] and its inability to form the tight complex with elastase [3,18,18,36]. In the present work, evidence for the oxidation of the two mets (Met351 and Met358) in the reactive center loop has been obtained by MS analysis of the proteolytic digest of the oxidized ␣1-PI. A third met, Met226, was also oxidized to a significant extent in the presence of bicarbonate. The results show that H2O2 oxidation of ␣1-PI can be accelerated by the addition of bicarbonate to the reaction medium (Fig. 3). At the highest concentration of bicarbonate used, the loss of inhibitory activity is essentially complete at the longest reaction time. The rate constants for the uncatalyzed pathway and the peroxymonocarbonate reaction are similar to those for free met and are consistent with acceleration of Met358 oxidation by formation of peroxymonocarbonate. Hypothetical pathway in neutrophil respiratory burst It is apparent that the previously unidentified HCO4⫺ pathway can make a substantial contribution to the overall oxidation rate for ␣1-PI by H2O2 in the presence of

Scheme 1.

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HCO3⫺. The results can be summarized in a model for the formation and reactivity of HCO4⫺ following superoxide, CO2, and elastase release in the respiratory burst [46,47] of neutrophils. In this hypothetical pathway, a portion of the H2O2 (and its highly nucleophilic conjugate base HO2⫺) produced by dismutation of superoxide is converted to HCO4⫺ via reaction with CO2 ([CO2] ⬇1 mM in normal lungs but likely to be locally much higher near neutrophils during reduced nicotinamide adenine dinucleotide phosphate-mediated burst [48]). The HCO4⫺ thus formed could react with the strongly nucleophilic exposed mets of ␣1-PI, thereby leading to the reduced inhibition of elastase by ␣1-PI and subsequent elastin tissue damage. Unlike indiscriminate radicals (such as OH), peroxides are selective oxidants and react more rapidly with the more potent nucleophiles available in the reaction mixture. Based on our estimates for the kinetic constants for perhydration of CO2, we also note that simultaneous release of CO2 and H2O2 by a cell could lead to a “sub-burst” of HCO4⫺ prior to the equilibration of Eqn. 1. The sub-burst would result from a more rapid perhydration rate of CO2 (left side of Fig. 1) compared to the hydration rate (right side of Fig. 1) after CO2 passes through the cell membrane into the surrounding medium. The magnitude of this sub-burst would depend upon the local concentrations of H2O2 and CO2. Spectroscopic and kinetic evidence for such a sub-burst of HCO4⫺ has been obtained in pH shock experiments using CO2/H2O2 solutions [14]. Although all the reactions (and many of the kinetic and thermodynamic parameters) in this model are known from our work, it is not known to what extent this pathway is responsible for oxidation of ␣1-PI in emphysema or neutrophil-based oxidative damage in other tissues [49]. The possibility of a role for peroxymonocarbonate depends on the local concentration gradients of HCO3⫺/CO2 around the activated neutrophil, because baseline plasma concentrations of HCO3⫺ (⬃25 mM) would have only a small catalytic effect (ca. 2-fold increase) on the oxidation (Fig. 3). Other oxidants known to be formed by activated neutrophils have been shown to be capable of oxidizing met to metSO, including the heterolytic oxidant OCl⫺ formed by myeloperoxidasecatalyzed oxidation of Cl⫺ by H2O2 [48]. Other possible biological roles Circulating met and metSO are found in plasma [50]. When bicarbonate ion is in large excess, the ratio of [HCO4⫺]:[H2O2] at equilibrium is ⬃0.3[HCO3⫺], and at typical bicarbonate levels in the blood (⬃0.02– 0.03 M) up to ⬃1% of H2O2 is converted to HCO4⫺ (or 0.01– 0.10 ␮M) [51,52]. At such a levels the oxidation rate of

met via a HCO4⫺ pathway would be up to twice that for H2O2 alone (although both oxidations would be slow given the low concentrations of the oxidants). Analogous models for the role of HCO4⫺ in other oxidative processes believed to involve H2O2 can be considered. In related studies, we have shown that bicarbonate accelerates the H2O2 oxidation of cysteine and related thiols [31]. Although the acceleration of peroxide oxidation of sulfides resulting from bicarbonate catalysis is relatively modest, the combination of bicarbonate and trace metals, particularly Mn(2⫹), strongly catalyzes the hydrogen peroxide oxidation of amino acids [53], linoleic acid [54], and guanosine [31], which are essentially unreactive with hydrogen peroxide and peroxymonocarbonate in the absence of metal catalysts. This oxidative chemistry may be particularly relevant in mitochondria, where a substantial amount of H2O2 is produced by the electron transport chain (⬃2% of O2 consumed [55,56]) and CO2 is generated in the matrix (primarily by the trichloroacetic acid cycle). The oxidative degradation of mitochondrial components has received much attention with respect to aging and disease [6]. It is also noteworthy that the peroxyanion HCO4⫺ can be considered a “trapped” form of peroxide in a cell or organelle, as it is not likely to cross membranes readily, as does neutral H2O2. Comparisons to peroxynitrite Peroxynitrite (ONOO⫺) and its conjugate acid (ONOOH) are also known to oxidize met residues [57], but the contrast between the effects of CO2 on ONOO⫺ and H2O2 chemistry is noteworthy. Carbon dioxide catalytically accelerates the decomposition of ONOO⫺ [58,59], and at physiological concentrations it almost completely inhibits oxidation of met residues by ONOO⫺ in vitro [57]. In contrast to HCO4⫺, which is stable for days in the presence of H2O2 and HCO3⫺, peroxynitrite has a short half-life (⬃2 s) around physiological pH values even in the absence of CO2 [59]. CONCLUSIONS

Physiological concentrations of HCO3⫺/CO2 can accelerate oxidation of met and protein mets by hydrogen peroxide. The relative biological importance of the H2O2/ HCO3⫺ system discussed here has yet to be established, but hypothetical roles in neutrophil respiratory burst and other biochemical processes should be investigated. The recent proposal that met is an intrinsic protein antioxidant [60] is also intriguing in light of our results. Is it possible that met residues are, in part, a defense against a pervasive ROS (HCO4⫺) that is formed from a common biological oxidant (H2O2) and the carbon waste product of aerobic metabolism (CO2/HCO3⫺)?

Methionine oxidation by peroxymonocarbonate Acknowledgements — This work was supported by a grant from the Army Research Office and the Edgewood Chemical Biological Center (ARO 37580-CH-2).

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