1398
Chem. Res. Toxicol. 1998, 11, 1398-1401
Kinetic Study of the Reaction of Glutathione Peroxidase with Peroxynitrite Karlis Briviba,† Reinhard Kissner,‡ Willem H. Koppenol,‡ and Helmut Sies*,† Institut fu¨ r Physiologische Chemie I, Heinrich-Heine-Universita¨ t Du¨ sseldorf, Postfach 101007, D-40001 Du¨ sseldorf, Germany, and Laboratorium fu¨ r Anorganische Chemie, Eidgeno¨ ssische Technische Hochschule, Universita¨ tsstrasse 6, CH-8092 Zu¨ rich, Switzerland Received April 27, 1998
Glutathione peroxidases and their mimics, e.g., ebselen or diaryl tellurides, efficiently reduce peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) to nitrite and protect against oxidation and nitration reactions. Here, we report the second-order rate constant for the reaction of the reduced form of glutathione peroxidase (GPx) with peroxynitrite as (8.0 ( 0.8) × 106 M-1 s-1 (per GPx tetramer) at pH 7.4 and 25 °C. The rate constant for oxidized GPx is about 10 times lower, (0.7 ( 0.2) × 106 M-1 s-1. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. The data support the conclusion that GPx can exhibit a biological function by acting as a peroxynitrite reductase.
Introduction -
1
Peroxynitrite/peroxynitrous acid (ONOO /ONOOH) is a strong oxidant and mediator of toxicity in inflammatory processes (1). ONOO- is relatively stable but upon protonation isomerizes rapidly to oxidize or nitrate target molecules via one or more strongly oxidizing intermediates. Low-molecular mass compounds such as cysteine, glutathione, ascorbate, or methionine react with peroxynitrite and can protect biomolecules against damage if present at sufficiently high concentrations. The secondorder rate constants for these reactions are in the range from 2 × 102 to 6 × 103 M-1 s-1 (for review, see ref 2). Natural organoselenium compounds such as selenomethionine and selenocystine or a synthetic organoselenium compound of pharmacological interest, ebselen, protects DNA from single-strand breaks and against oxidation and nitration reactions about 100-1000-fold more effectively than their sulfur analogues and other low-molecular mass compounds, such as cysteine, glutathione, or methionine (3). Furthermore, ebselen, a mimic of glutathione peroxidase, reacts very rapidly with ONOO- (4), with a second-order rate constant of 2 × 106 M-1 s-1 (5). There are few other compounds that react with similarly high rate constants, e.g., Fe and Mn porphyrins (6, 7). Recently, we have shown that glutathione peroxidase (GPx2), a tetrameric enzyme containing selenocysteine at the active center of each subunit (8), reduces peroxynitrite to nitrite and protects against oxidation and nitration reactions (9). Furthermore, GPx acts as a peroxynitrite reductase in the presence of thiols in a * Address for correspondence: Institut fu¨r Physiologische Chemie I, Heinrich-Heine-Universita¨t Du¨sseldorf, Postfach 101007, D-40001 Du¨sseldorf, Germany. Telephone: +49-211-811-2707. Fax: +49-211811-3029. † Heinrich-Heine-Universita ¨ t Du¨sseldorf. ‡ Eidgeno ¨ ssische Technische Hochschule. 1Systematic names: ONOO-, oxoperoxonitrate(1-); ONOOH, hydrogen oxoperoxonitrate; NO•, nitrogen monoxide, also known as nitric oxide. 2Abbreviations: GSH, glutathione; GPx, glutathione peroxidase.
catalytic reaction with the stoichiometry of two thiols consumed per one peroxynitrite reduced, i.e., the “classical” GPx reaction (9). Therefore, the study of selenoproteins in terms of their reaction with peroxynitrite is of interest. Here, we used the stopped-flow technique to study the reaction of GPx with peroxynitrite.
Experimental Procedures Reagents. Glutathione peroxidase from bovine erythrocytes (MW of 21.9 kDa per subunit) and manganese dioxide (MnO2) were from Fluka Chemie AG (Buchs, Switzerland). Glutathione and glutathione disulfide reductase were from Sigma (Deisenhofen, Germany). NADPH was from Boehringer (Mannheim, Germany). Other chemicals and solvents were from Merck (Darmstadt, Germany). Redox State of Glutathione Peroxidase. Iodoacetate and cyanide were used as probes to characterize the redox state of the selenocysteine in the active center of GPx. The reduced form of GPx contains the selenol, which is carboxymethylated by iodoacetate (10). In contrast, the oxidized enzyme, present as the selenosulfide with glutathione (ESe-SG) or as the selenenic acid (ESe-OH), does not react with iodoacetate. These oxidized forms can be readily reduced back to the selenol by thiols, as can be verified by their catalytic activity to reduce hydroperoxides to the corresponding alcohols in the presense of thiols. Using cyanide, one can distinguish between ESe-SG and ESeOH; whereas ESe-SG is inactivated by cyanide, ESe-OH is not (11). To evaluate the reduction state of selenium in the GPx preparations, GPx (0.2 µM) was incubated with iodoacetate (1 mM) for 20 min, or with potassium cyanide (10 mM), or alone for 120 min in 0.1 M potassium phosphate buffer at pH 7.5 and 25 °C as described previously (11). Small aliquots (10 µL) taken at zero time and after incubation were analyzed for GPx activity (see below). Preparation of the Reduced and Oxidized Form of Glutathione Peroxidase. “Reduced GPx” was prepared by incubation of the enzyme (50 µM GPx) with 2-mercaptoethanol (5 mM) in 2 mL of phosphate-buffered saline [PBS; 137 mM sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogen phosphate, and 2 mM potassium dihydrogen phosphate (pH 7.4)] for 30 min at room temperature and then washed twice with 12 mL of PBS in a centrifugal protein
10.1021/tx980086y CCC: $15.00 © 1998 American Chemical Society Published on Web 12/03/1998
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Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1399
concentrator (MW cutoff of >10 kDa; Millipore, Eschborn, Germany). Distilled water and 0.2 M phosphate buffer were added to GPx to obtain a final concentration of phosphate buffer of 0.1 M. This preparation of GPx was inactivated by iodoacetate by >90%, but not by cyanide, indicating that the enzyme was in the reduced form. “Oxidized GPx” was prepared by treatment of GPx with H2O2 at a molar ratio of thiols in the GPx preparation to hydrogen peroxide of 1:1 for 30 min at room temperature and then washed with 12 mL of PBS in the centrifugal protein concentrator. No detectable changes in activity of GPx were found using tert-butyl hydroperoxide as a substrate. The preparation of oxidized GPx was stable for more than 2 h when incubated at 25 °C and was insensitive to treatment with iodoacetate or cyanide, indicating that oxidation of selenol to selenenic acid had taken place. The protein concentration was determined with the Bio-Rad protein assay. Synthesis of Peroxynitrite. Peroxynitrite was synthesized from potassium superoxide and nitric oxide as described previously (13), and hydrogen peroxide was eliminated by passage of the peroxynitrite solution over MnO2 powder. The peroxynitrite concentration was determined spectrophotometrically at 302 nm ( ) 1705 M-1 cm-1) (14). Kinetic Analysis. The kinetic study was carried out as described previously (5) on an Applied Photophysics SX17MV stopped-flow apparatus using Spektrakinetic software (Leatherhead, U.K.). Experiments were performed at 25 °C. Stoppedflow measurements were carried out at 302 nm by mixing GPx (10-28 µM per GPx subunit) with peroxynitrite (5 µM) dissolved in 0.1 M phosphate buffer and in 0.01 M aqueous sodium hydroxide, respectively, at a ratio of 1:1. GPx was used in 2-6fold excess over peroxynitrite. Under these conditions, the formation of an adduct between peroxynitrite and peroxynitrous acid is avoided (15). At least 10 kinetic traces were recorded and averaged for each determination. After the stopped-flow experiment at pH 7.4, GPx and decay products were collected, concentrated, and reduced by mercaptoethanol as described above. The activity of GPx as a peroxidase was also monitored using tert-butyl hydroperoxide as a substrate. GSH Peroxidase Assay. GPx activity was followed spectrophotometrically at 340 nm as described in refs 8 and 16 with minor modifications. The test mixture contained GSH (1 mM), diethylenetriaminepentaacetic acid (1 mM), glutathione disulfide reductase (0.6 unit/mL), and NADPH (0.1 mM) in 0.1 M sodium phosphate (pH 7.3). GPx samples were added to the test mixture at room temperature, and the course of NADPH oxidation was followed spectrophotometrically for 2 min. The reaction was started by the addition of tert-butyl hydroperoxide (1.2 mM). Activity was calculated from the rate of NADPH oxidation.
Figure 1. Stopped-flow detection of peroxynitrite disappearance in the presence of glutathione peroxidase (GPx). (A) Peroxynitrite (5 µM) in 0.01 M NaOH and reduced GPx (16 µM, per subunit) in 100 mM phosphate buffer were mixed (1:1) at 25 °C. (B) Similar experiment with oxidized GPx (26 µM). The reactions were followed at 302 nm; the pH after mixing was 7.4. The curves are the average of 10 traces. Under the same conditions, peroxynitrite alone takes several seconds to decay spontaneously.
Results Figure 1 shows the disappearance of peroxynitrite as measured at 302 nm at pH 7.4 and 25 °C. The reaction of peroxynitrite with reduced GPx and with oxidized GPx is shown in panels A and B of Figure 1, respectively. The decrease in absorbance follows pseudo-first-order kinetics in both cases, and the amplitude is about 4 milliabsorbance units, which is the expected decrease of absorbance due to decomposition of 2.5 µM peroxynitrite. The pseudo-first-order rate constants for peroxynitrite decomposition are shown as a function of the concentration of reduced GPx (b) in Figure 2. The observed secondorder rate constant for this reaction is estimated to be (2.0 ( 0.2) × 106 M-1 s-1, expressed per monomer of GPx; per concentration of tetrameric enzyme, it is four times higher (8.0 × 106 M-1 s-1). Further stopped-flow experiments were carried out by mixing peroxynitrite with a mixture of GPx and GSH.
Figure 2. Pseudo-first-order rate constants for peroxynitrite disappearance as a function of glutathione peroxidase concentration. Experimental conditions were like those described in the legend of Figure 1. Reduced GPx (b), oxidized GPx (O), or GPx and GSH (2) were mixed (1:1) to give the indicated final concentrations (per subunit GPx). The GSH concentration was 0.5 mM.
GSH (0.5 mM) alone had only a small effect on the rate of disappearance of peroxynitrite; the decay rate of peroxynitrite in the presence of 0.5 mM GSH was 0.7 s-1. The pseudo-first-order rate constants for peroxynitrite disappearance versus GPx concentration in the presence of a constant concentration of GSH at 0.5 mM are shown
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Table 1. Second-Order Rate Constants for the Reaction of Peroxynitrite with Some Biological Molecules and the Rates of Disappearance of Peroxynitrite in the Presence of These Compounds at in Vivo Concentrations second-order rate constant (M-1 s-1) pH T (°C) spontaneous decay protein glutathione peroxidase reduced oxidized oxidized hemoglobin low-molecular mass compound CO2 glutathione ascorbate a
(8.0 ( 0.8) × 106 (7.4 ( 2.0) × 105 (1.8 ( 0.8) × 105 2.5 × 104 3× 5.8 × 102 104
50
ref
7.4
25
1
7.4 7.4 7.1 7.4
25 25 37 37
this paper this paper 28 31
in vivo concentration (M)
ref
rate of disappearance of peroxynitrite (s-1) 0.4
7.4 7.4
25 25
32 6
7.4
25
34
1.9 × 10-6 (liver)
30a
5 × 10-3 (erythrocytes) 10-3
1× (plasma) 10 × 10-3 (liver) 2 × 10-6 (plasma) 1.4 × 10-2 (neutrophils) 7 × 10-5 (plasma)
15.2 125
32 23 33 35 36
30 5.8 0.002 0.7 0.004
The GPx concentration was calculated from the amount of selenium in liver and the 23% contribution to GPx (37).
in Figure 2 (2). The second-order rate constant is estimated to be (9.2 ( 0.9) × 106 M-1 s-1 per tetramer of GPx. We recently demonstrated that reduced GPx protects more effectively against peroxynitrite-mediated nitration or oxidation reactions than oxidized GPx (9). The pseudofirst-order rate constants as a function of oxidized GPx concentration are also shown in Figure 2 (O). The secondorder rate constant for oxidized GPx is (0.74 ( 0.2) × 106 M-1 s-1 (per GPx tetramer). The GPx was collected after the stopped-flow experiments in Figure 1A, and no detectable changes in activity of GPx were found when 5-10 µM reduced GPx was mixed with 2.5 µM peroxynitrite in the stopped-flow apparatus; the activity of GPx before and after mixing with peroxynitrite was 29.3 ( 0.5 and 28.2 ( 0.6 units/ mg of protein, respectively.
higher than that for GSH, but 2-fold lower than that calculated for CO2. Although CO2 provides a major pathway for the disappearance of peroxynitrite, GPx could play a significant role in the defense against peroxynitrite in vivo. Locally, the concentration of GPx may be higher than 1.9 µM; e.g., the membrane-bound forms of GPx and, consequently, the disappearance via GPx would be more important in these compartments. These considerations need to be extended; for example, the regeneration of GPx after the peroxynitrite-dependent step depends on the rate of reaction by GSH, for which the value of about 2 × 106 M-1 s-1 (8) is quite substantial. Given a concentration of about 10 mM GSH in the cell (23) and the GSH:GSSG ratio being about 300 (24), the reduced rather than oxidized state of GPx is thermodynamically favored. The reaction of peroxynitrite with reduced GPx, but not with oxidized GPx, is therefore of physiological interest.
Discussion
Peroxynitrite can inactivate a number of enzymes such as aconitase (25) or alcohol dehydrogenase (17). Even the precursor of peroxynitrite, nitric oxide, is capable of inactivating GPx (26), oxidizing selenocysteine with the formation of selenenyl sulfide with a cysteine residue of GPx (27); this may occur directly or through the formation of nitrogen dioxide or dinitrogen trioxide. To test whether the activity of GPx is lowered under conditions used in this study, we estimated the GPx activity using tert-butyl hydroperoxide as the substrate, and no detectable changes in the activity of GPx were observed, as described above. This is in line with our report that exposure of 150 nM GPx in phosphate buffer at pH 7.3 to a bolus addition of peroxynitrite up to 30 µM did not detectably change the capability of GPx to reduce tertbutyl hydroperoxide (9).
The second-order rate constant for the reaction of reduced GPx with peroxynitrite was estimated to be 8.0 × 106 M-1 s-1 (per tetramer of GPx, at pH 7.4 and 25 °C). This rate constant is higher than that observed for other proteins, e.g., alcohol dehydrogenase (17), albumin (18), or lactoperoxidase (19), and it is similar to that reported for myeloperoxidase (19). Furthermore, GPx reacts about 2-4 orders of magnitude faster with peroxynitrite than CO2, ascorbate, or thiols, e.g., glutathione, at pH 7.4 (Table 1). For homogeneous systems, multiplication of the concentration of a given compound with the corresponding rate constant for the reaction of peroxynitrite with that compound yields the rate of disappearance of peroxynitrite. This approach was used previously to roughly estimate the biological relevance of the reaction of peroxynitrite with CO2 (20), hemoglobin, and peroxidases (21), although concentrations in biological systems are difficult to determine. With regard to cellular systems, hemoglobin has a high capacity to react with peroxynitrite in erythrocytes (Table 1). However, hemoglobin is not able to protect the plasma membrane of erythrocytes when peroxynitrite is generated outside the cells; it has been reported that peroxynitrite effectively induces hemolysis (22). The rates of disappearance of peroxynitrite calculated using in vivo concentrations of some biological molecules such as GSH and CO2, which have been proposed as major targets for peroxynitrite, are shown in Table 1. The rate for GPx is about 2-fold
Recently, a second-order rate constant of 1.8 × 105 M-1 s (per GPx tetramer) was reported for the reaction of GPx with peroxynitrite at pH 7.1 (28). This rate constant is about 4-fold lower than that determined in this paper for oxidized GPx (7.4 × 105 M-1 s-1 at pH 7.4) and is substantially lower than the value of 8.0 × 106 M-1 s-1 for reduced GPx. We have shown that the reduced, but not the oxidized, form of GPx effectively protects against oxidation and nitration reactions caused by peroxynitrite (9). The fast reaction of GPx with peroxynitrite supports our proposal that GPx and other selenoproteins carry out a biological function in the defense against peroxynitrite (9, 29). -1
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Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1401
Acknowledgment. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 503, Project B1, and by the ETH Zuerich.
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