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Feb 16, 2005 - Dismutase Stimulates Bicarbonate-Dependent. Peroxidase Activity via Formation of a Carbonate. Radical. Chandran Karunakaran, Hao Zhang,...
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Chem. Res. Toxicol. 2005, 18, 494-500

Thiol Oxidase Activity of Copper, Zinc Superoxide Dismutase Stimulates Bicarbonate-Dependent Peroxidase Activity via Formation of a Carbonate Radical Chandran Karunakaran, Hao Zhang, Joy Joseph, William E. Antholine, and B. Kalyanaraman* Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received September 9, 2004

Here, we investigated the effect of bicarbonate anion (HCO3-) on the peroxidase activity stimulated by the thiol oxidase activity of copper, zinc superoxide dismutase (SOD1) using electron spin resonance (ESR) and optical techniques. Low temperature direct ESR revealed that cysteine (Cys) caused the reduction of copper(II) to copper(I) that was reoxidized by molecular oxygen to copper(II) at the active site of SOD1. The addition of HCO3- to aerobic incubations containing SOD1, Cys, and DTPA in phosphate buffer enhanced the peroxidase activity of SOD1, as measured by hydroxylation of cyclic nitrone spin traps, dichlorodihydrofluorescein oxidation to dichlorofluorescein, and oxidation of tyrosine to dityrosine. The addition of catalase inhibited the SOD1 peroxidase activity stimulated by the thiol oxidase actvity, implicating an intermediary role for H2O2 in SOD1/Cys/HCO3--mediated oxidation and hydroxylation reactions. Using a competitive kinetic method, rate constants for the reaction between the oxidant formed in the SOD1/Cys/HCO3- system and selected inhibitors were measured. On the basis of these rate constants, we conclude that the thiol oxidase activity of SOD1 stimulates carbonate anion radical (CO3•-) formation in the presence of HCO3- and that the CO3•- formed in the SOD1/Cys/ HCO3- system is responsible for oxidation and hydroxylation reactions. Biological implications of this finding are discussed.

Introduction Recently, a new activity for the enzyme copper, zinc superoxide dismutase (SOD1) in the presence of cysteine (Cys) was discovered (1). At low concentrations, SOD1 catalyzed the autoxidation of thiols via a thiol oxidase activity (1). Cys is a naturally occurring amino acid that is present at a concentration of ∼250 µM in the brains of normal human subjects (2). Its concentrations under pathological conditions have not been well-defined. Cys is also a more potent excitotoxin than β-N-methylaminoL-alanine and has been implicated in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson’s and Alzheimer’s diseases (35). Elevated levels of Cys to sulfate ratios have been found in patients suffering from such neurological diseases (4). L-Cys was reported to be a bicarbonate (HCO3-) sensitive endogenous excitotoxin (3). The excitotoxic potency of Cys increased in the presence of physiologically relevant concentrations of HCO3- (3). The HCO3- effect on Cys-induced neurotoxicity was shown to be independent of pH changes (4). The toxic gain in function of SOD1 in ALS had been attributed to its HCO3--dependent peroxidase activity (6). Previously, it has been reported that HCO3- enhanced the SOD1 peroxidase activity via * To whom correspondence should be addressed. Tel: 414-456-4035. Fax: 414-456-6512. E-mail: [email protected].

a mechanism involving the carbonate anion radicals (CO3•-) (7-10). L-Cys-induced excitotoxicity had been linked to formation of oxy radicals (4). Thus, it is of interest to investigate the mechanism of generation of oxidants during SOD1-catalyzed oxidation of Cys in the presence of HCO3-. Using the direct electron spin resonance (ESR) and spin trapping techniques, we show that HCO3- stimulates the SOD1 peroxidase activity in the presence of Cys in a concentration-dependent manner. We postulate that HCO3- accelerates the thiol oxidase activity via formation of CO3•-. The second-order rate constants determined for the radical species generated by SOD1/ Cys/ HCO3- in the presence of various agents are consistent with the literature rate constants reported for the CO3•- radical.

Experimental Procedures Materials. SOD1, carbonic anhydrase from bovine erythrocytes, and catalase from beef liver were obtained from Roche Diagnostics. Cys, tyrosine, tryptophan, glutathione, methionine, phenylalanine, histidine, uric acid, sodium ascorbate, NADPH, hydrogen peroxide, sodium bicarbonate, sodium formate, sodium azide, ethanol, Me2SO, sodium hydrogen phosphate, trizma base, and DTPA were purchased from Sigma (St. Louis). 5,5′Dimethyl-1-pyrroline N-oxide (DMPO) obtained from Sigma was treated with activated charcoal to remove the colored impurities, and its concentration was determined spectrophotometrically at 227 nm using an extinction coefficient of 8000 M-1 cm-1 (11).

10.1021/tx049747j CCC: $30.25 © 2005 American Chemical Society Published on Web 02/16/2005

HCO3- Accelerates SOD1/Thiol Oxidase-Mediated Activity BMPO was synthesized as described previously (12). R-(4Pyridyl-1-oxide)-N-tert-butyl nitrone (POBN) and 3,5-dibromo4-nitrosobenzenesulfonic acid (DBNBS) obtained from Sigma were used as received. DCFH was purchased from Molecular Probes, Inc. (Eugene, OR). ESR Measurements. A typical reaction mixture (200 µL) for ESR measurements consisted of SOD1 (30 µM), thiols (0.1-1 mM), and HCO3- (25 mM) in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 µM). The X-band ESR of copper and nitroxide spin adducts was recorded on a Bruker E500 ELEXYS spectrometer with 100 kHz field modulation equipped with an Oxford Instruments ESR-9 helium flow cryostat and a DM_0101 cavity. Incubation mixtures (200 µL) containing SOD1 and Cys were transferred to a 4 mm quartz ESR tube (Wilmad) and frozen immediately in liquid nitrogen for ESR analysis. ESR spectral parameters were extracted from the spectra using the QPOWA program based on the successful fitting to the following orthorhombic spin Hamiltonian (eq 1) (13).

H ) β(gz Sz Bz + gx Sx Bx + gy Sy By) + Az Iz Sz + Ax Ix Sx + Ay Iy Sy (1)

Chem. Res. Toxicol., Vol. 18, No. 3, 2005 495 spin trapping with DMPO (16, 17). At saturating concentrations of DMPO, the CO3•- radical formed is consumed through eqs 3 and 4. kDMPO

8 DMPO-OH DMPO + CO3•- 9 H O 2

kinhibitor

inhibitor + CO3•- 98 products

(4)

d[DMPO-OH]/dt ) kDMPO [DMPO][CO3•-]

(5)

In the presence of an inhibitor at a saturating level of DMPO, the rate of spin trapping is equal to the rate of carbonate anion radical generation, d[CO3•-]/dt.

d[CO3•-]/dt ) kDMPO [DMPO][CO3•- ] + kinhibitor [inhibitor][CO3•-] (6) Dividing eq 6 by eq 5, eq 7 is obtained.

d[CO3•-] /dt where β is the Bohr magneton and gx, gy, gz and Ax, Ay, Az are the components with respect to the principal axes of the g-tensor and the hyperfine tensor. Spectrometer conditions were as follows: microwave frequency, 9.63 GHz; modulation frequency, 100 kHz; modulation amplitude, 5 G; receiver gain, 85 dB; time constant, 0.01 s; conversion time, 0.08 s; sweep time, 83.9 s; and microwave power, 100 µW. ESR Spin Trapping Analysis. A typical reaction mixture (∼100 µL) for ESR experiments consisted of SOD1 (30 µM), DMPO (50 mM), Cys (500 µM), and HCO3- (25 mM) in a phosphate buffer (100 mM, pH 7.4) containing DTPA (0.1 mM). ESR spectra were recorded at room temperature on a Bruker EMX spectrometer operating at 9.859 GHz with a 100 kHz field modulation and equipped with a TE102 cavity. Spectrometer conditions were as follows: modulation amplitude, 1 G; time constant, 0.00512 s; conversion time, 0.01024 s; sweep time, 10.49 s; and microwave power, 20 mW. Time-course ESR spin trapping spectra were obtained by recording 60 consecutive spectra using a scan time of 40 s. ESR data analysis was written using the Microsoft Visual C++. This program allows direct access to Bruker EMX spectrometer three-dimensional (3D) data files and incorporates the singular value decomposition (SVD) algorithm from Numerical Recipes in C, as reported previously (14). SVD was performed on a matrix m × n, A constructed from the 3D ESR data sets (intensity vs magnetic field vs time) with n rows of spectral data points and m columns of delay time points, respectively. In this study, m was 60 and n was 1024. The matrix A was converted to an n × n diagonal matrix, W, by means of an m × n orthogonal matrix, U, and an n × n orthogonal matrix, V, through the following equation

A)UWV

(2)

This operation thus decomposes the data set into principal spectral components obtained from the diagonal elements of W, called singular values of A, and arranges them in the order of magnitude. The number of spectral components indicates the number of kinetically independent processes within the data, and principal spectral components each represent a linear combination of the underlying spectral species. Noise reduction was achieved by reconstructing the data set using only the principal components and discarding the components that contain only noise. Spectral simulations were performed using the WINSIM program (NIEHS, NIH, Research Triangle Park, NC) (15). Rate Constant Determinations. The second-order rate constants for reactions between the radical species formed in SOD1/Cys/HCO3- and other inhibitors were measured using

(3)

d[DMPO-OH] /dt

)1+

kinhibitor [inhibitor] kDMPO [DMPO]

(7)

If Ro and R represent the initial rates of spin trapping in the absence and presence of inhibitors, respectively, eq 7 may then be rewritten as follows:

kinhibitor [inhibitor] Ro )1+ R kDMPO [DMPO]

(8)

For ESR kinetic measurements, the concentration of DMPO (100 mM) was fixed while the inhibitor concentrations were varied. The formation of the DMPO-OH adduct was monitored for a period of 60 min. The initial rate of DMPO-OH formation was used in this calculation, so as to minimize the decomposition of the spin adduct. Measurement of Thiol Oxidase-Stimulated Bicarbonate-Dependent Peroxidase Activity. DCFH diacetate was hydrolyzed freshly in every experiment according to the published procedure (18). Briefly, DCFH was dissolved in methanol and hydrolyzed by NaOH (0.01 M) in the dark for 30 min at room temperature and stored in an ice bath during the experiment. SOD1 thiol oxidase activity via peroxidase activity was measured as follows. SOD1 (15 µM) was mixed with a solution of DCFH (40 µM) in a phosphate buffer (0.1 M, pH 7.4) containing DTPA (100 µM), Cys (0-1 mM), and various amounts of sodium bicarbonate (0-50 mM). The reaction was initiated by adding either Cys or HCO3-, and the formation of DCF was monitored at 505 nm. DCF formation was also measured fluorometrically using the excitation and emission wavelengths, 495 and 520 nm, respectively (18). In control samples where DCFH was omitted, an equal volume of methanol/0.01 N NaOH/ phophate buffer (1:4:20 v/v/v) was added. Fluorescence Measurements. Fluorescence experiments were performed on a Shimadzu RF-5301 PC spectrofluorometer (Shimadzu Scientific Instruments Inc.). Spectra were obtained at the indicated excitation and emission wavelengths using a 5 and 10 mm slit width, respectively.

Results Changes in the ESR and Optical Spectra of SOD1 in the Presence of Cys and HCO3-. Time-dependent EPR spectra obtained from incubations containing SOD1 and Cys in the presence and absence of HCO3- are shown in Figure 1A, B. The computer simulations shown in dotted lines are based on the spin Hamiltonian parameters of two species, namely, the native enzyme (gx ) 2.030, gy ) 2.090, gz ) 2.268, and Az ) -141 G) and a

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Karunakaran et al. Scheme 1. Reaction Scheme Based on Kinetic Simulations for the HCO3--Accelerated Thiol Oxidase Activity of SOD1a

a Rate constants used to simulate the loss and reappearance of E-Cu(II) in incubation containing SOD1 (100 µM), HCO3- (25 mM), and Cys (500 µM) are as follows: k1, 1.8 M-1 s-1; k2, 12.5 M-1 s-1; k3, 2 × 109 M-1 s-1; k4, 60 M-1 s-1; k5, 20 M-1 s-1; k6, 3.5 × 109 M-1 s-1; k7, 10-2 M-1 s-1; k8, 5 × 10-2 M-1 s-1; k9, 2.6 × 109 M-1 s-1; and k10, 107 M-1 s-1.

Figure 1. Time-dependent changes in ESR spectra of SOD1 in the presence of Cys and HCO3-. (A) Time-dependent changes in ESR of SOD1-Cu(II) (30 µM) in the presence of Cys (500 mM) in phosphate buffer (100 mM, pH 7.4) containing 100 µM DTPA. (B) Same as panel A but in the presence of HCO3- (25 mM). (C) Same as panel A but in the presence of catalase (10 µg/mL). (D) Same as panel B but with catalase (10 µg). Incubation mixtures were transferred to a quartz ESR tube at the indicated time point, and spectra were recorded at 8 K. The solid line is the experimental spectrum, and the dotted line is the simulated spectrum according to the parameters presented in the Results section. Spectrometer conditions are described in the Experimental Procdures. Spectra were recorded at 10 K. (E) The change in the ESR signal intensity monitored at the position indicated by an arrow in panels A-D and F. Optical spectra obtained under similar conditions described in panels A-C at room temperature except that the concentration of SOD1 was 100 µM. Optical spectra were recorded immediately after the addition of Cys to incubation mixtures containing SOD1 in phosphate buffer (pH 7.4, 100 mM) and other components described in panels A-D. Theoretical simulations (solid line) were performed using the rate constants indicated in Scheme 1.

modified less rhombic form of the enzyme (gx ) 2.045, gy ) 2.055, gz ) 2.268, and Az ) -150 G). In the presence of HCO3-, the spectral parameters due to the native enzyme prevailed, whereas in the absence of HCO3- the spectral parameters are mostly indicative of the less rhombic form of the enzyme. This suggests that HCO3partially protected the Cu(II) active site from Cysmediated oxidative damage. This analysis is consistent with our previous interpretations using the SOD1/H2O2 system (19). The changes in the Cu(II) EPR signal intensity in the g⊥ region are shown in Figure 1E. As shown in Figure 1, the Cu(II) signal intensity slowly decreased up to 45 min due to Cys-dependent reduction of Cu(II) to Cu(I) and then increased due to reoxidation of Cu(I) to Cu(II) by molecular oxygen (Figure 1E, trace A). However, in the presence of HCO3-, the Cu(II) signal intensity grew back

more rapidly, presumably due to reoxidation of Cu(I) by the oxidant generated from HCO3- (Figure 1E, trace B). Similar spectral changes were reported for the bovine SOD1/H2O2/HCO3- system (19). In the presence of catalase (Figure 1C), the reappearance of copper(II) signal intensity was considerably faster (Figure 1E, trace D), implicating an oxidation/reduction reaction between Cu2+ and H2O2. The effect of Cys on the reduction of copper(II) in SOD1 was also monitored by following absorbance at 680 nm (Figure 1F). Similar to the ESR profile of Cu(II), the absorbance initially decreased up to 15 min and increased slowly thereafter. The optical results (Figure 1F) are in close agreement with ESR results (Figure 1E). The solid line in Figure 1F represents the simulation of the experimental data (open circle) using the kinetic model shown in Scheme 1. Bicarbonate Stimulates Oxidation and Hydroxylation of Cyclic Nitrone Spin Traps in the Presence of SOD1 and Cys: Intermediacy of CO3•- Radical but not •OH. The effect of the Cys on the kinetics of formation of DMPO-OH from incubations containing SOD1 and HCO3- in phosphate buffer is shown in Figure 2A. In the absence of Cys, no DMPO-OH was formed. With increasing Cys, the DMPO-OH formation rapidly increased (Figure 2A,B). However, at higher concentrations of Cys (1 mM), the DMPO-OH decreased possibly due to the reduction of DMPO-OH by excess Cys. The effect of the HCO3- concentrations on the time-course of DMPO-OH formation is shown in Figure 2C,D. In the absence of HCO3-, no DMPO-OH was formed. However, addition of HCO3- to the incubation mixture induced the DMPO-OH formation that increased with increasing HCO3- up to 25 mM. Recently, Liochev and co-workers (20) had reported that CO2, but not HCO3-, enhanced the peroxidase activity of SOD1. Figure 2E shows the effect of SOD1 on DMPO-OH formation. No ESR signal due to DMPO-OH was detected in the absence of SOD1. With increasing SOD1, the DMPO-OH adduct increased in a concentration-dependent manner (Figure 2F). Figure 2G shows the effect of varying phosphate levels on DMPOOH formation. There was an inverse dependence of the phosphate ion (in the concentration range of 25-250 mM)

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Figure 3. Effect of hydroxyl radical scavengers on thiol oxidase/HCO3-/SOD1 peroxidase-induced formation of DMPO adducts. Incubations contained bovine SOD1 (30 µM), Cys (500 µM), HCO3- (25 mM), and DMPO (50 mM) in phosphate buffer (100 mM, pH 7.4) containing 100 µM DTPA and catalase (10 µg/mL) or typical hydroxyl radical scavengers EtOH (2 vol %) (control), in the presence of formate (50 mM) or in the presence of azide (50 mM). Spectra were obtained within 10 min after adding all of the components.

Figure 2. Kinetics of formation of DMPO-OH by thiol oxidase/ bicarbonate dependent SOD1 peroxidase activity. Incubations contained different concentrations of bovine SOD1 (30 µM), Cys (500 µM), HCO3- (25 mM), and DMPO (50 mM) in phosphate buffer (100 mM, pH 7.4) containing 100 µM DTPA. (A) The steady state ESR spectra measured after 20 min with increasing concentrations of Cys (a-f). (B) The time-dependent increase in the formation of DMPO-OH obtained by monitoring the lowfield line of DMPO-OH with increasing concentrations of Cys (a-f). (C) The same as panel A but with increasing concentrations of bicarbonate (a-f). (D) The same as panel B but with increasing concentrations of bicarbonate (a-f). (E) The same as panel A but with increasing concentrations of SOD1 (a-f). (F) The same as panel B but with increasing concentrations of SOD1 (a-f). (G) The same as panel A but with increasing concentrations of phosphate (a-e). (H) The same as panel B but with increasing concentrations of phosphate (a-f).

on DMPO-OH formation (Figure 2H). This suggests that the competition between HCO3- and phosphate ion at the Cu(II) active site modulates the SOD1 peroxidase activity stimulated by the thiol oxidase activity. The addition of catalase to the incubation mixture abolished the formation of DMPO-OH adduct (Figure 3), suggesting that H2O2 formed from SOD1 thiol oxidase activity stimulated HCO3--dependent SOD1 peroxidase activity. In the presence of ethanol, the ESR spectrum (RN ) 15.8 G, RH ) 22.8 G) due to the DMPO-Et adduct (marked 9) was detected from incubations containing SOD1, Cys, HCO3-, and DMPO (Figure 3A). The DMPOEt adduct was most likely formed from trapping of the hydroxyethyl radical formed from the abstraction of a hydrogen atom from ethanol by CO3•- (7). In the presence of formate, the SOD1/Cys/ HCO3- system yielded the ESR spectrum due to the DMPO-CO2- adduct (RN ) 15.6 G, RH ) 18.7 G) (marked D) (7). The formate radical anion (CO2•-) is presumably formed from oxidation of HCO2-

by CO3•- (7). The addition of the azide anion to the incubation mixture containing SOD1, Cys, and HCO3resulted in the formation of the DMPO-azide adduct (DMPO-N3-) spectrum (RN ) 14.7 G, RH ) 14.7 G, and RNβ ) 3.1 G) (marked 2). The DMPO-N3- adduct is formed from trapping of the azide radical (by DMPO) resulting from the one-electron oxidation of azide by CO3•-. Replacement of SOD1 with Cu2+ (30 µM) did not generate these DMPO adducts (data not shown). This result clearly suggests that an oxidant formed from the oxidation of HCO3- by the SOD1/Cys system, not from free Cu2+ ions, is responsible for the hydroxylation of DMPO. On the basis of previous studies (7, 9), we attribute the oxidant generated in this system to be the carbonate radical anion (CO3•-). Previously, we showed that DMSO, by virtue of its differential reactivity with CO3•- and •OH, can be used to distinguish between CO3•- and •OH formation in the SOD1/H2O2/HCO3- system (7). Therefore, we investigated the effect of DMSO during HCO3--dependent SOD1/thiol oxidase-mediated SOD1 hydroxylation/oxidation of several nitrones. The ESR spectra obtained from the incubation mixtures containing SOD1, Cys, DMPO, BMPO or POBN, HCO3- , and DTPA in a phosphate buffer with and without DMSO are shown in Figure 4A-F. In contrast to the cyclic nitrone traps, DMPO and BMPO, which form persistent hydroxylated adducts (Figure 4A,C), the hydroxylated adduct of the open chain nitrone, POBN, decomposed to form a secondary radical adduct (Figure 4E). The ESR spectra consisted of two adducts corresponding to POBN-OH and the N-tert-butyl hydronitroxide, MNP-H. We propose that POBN-OH decomposed to the aldehyde and MNP-hydroxylamine, which was further oxidized by CO3•- to the MNPhydronitroxide (RN ) 14.4 G, RH ) 14.4 G). The addition of DMSO, a frequently used hydroxyl radical scavenger, had no effect on the ESR signal intensity of the hydroxylated spin adducts (Figure 4A,C,E).

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Figure 4. Effects of DMSO on Cys/HCO3-/SOD1 and Fe2+/ H2O2-generated nitrone-derived spin adducts. (A, top) The steady state ESR spectrum of DMPO-OH generated from incubations containing SOD1 (30 µM), Cys (500 µM), HCO3(25 mM), and DMPO (50 mM) in phosphate buffer (pH 7.4, 100 mM) containing DTPA (100 µM) and (bottom) same as above but in the presence of DMSO. (B, top) The ESR spectrum of DMPO adducts formed from the interaction between Fe(II) and H2O2 in phosphate buffer and (bottom) same as above but with DMSO. (C, top) Same as panel A except that the incubation mixtures contained the spin trap BMPO (75 mM) and (bottom) same as above but with DMSO. (D, top) The ESR spectrum of BMPO adducts formed from the interaction between Fe(II) and H2O2 in phosphate buffer (pH 7.4, 100 mM) and (bottom) same as above but with DMSO. (E, top) same as panel A except that the incubation mixtures contained POBN (75 mM) and (bottom) same as above but with DMSO. (F, top) The ESR spectra of POBN adducts formed from the interaction between Fe(II) and H2O2 in phosphate buffer and (bottom) same as above but with DMSO.

In contrast to the SOD1/Cys/HCO3- system, the addition of DMSO to a mixture containing Fe2+ and hydrogen peroxide (the Fenton system) diminished the ESR spectrum of the hydroxylated adduct, along with a concomitant increase in the spectral intensity due to the methyl radical adduct (Figure 4B,D,F). For example, with POBN, the ESR spectrum of the hydroxylated adduct was replaced by the POBN-methyl adduct. From these results, we conclude that free hydroxyl radicals are not generated in the SOD1/Cys/HCO3- system and that the ESR spin trapping technique can be used to monitor HCO3--dependent Cys-mediated peroxidase activity of SOD1. Thiol Oxidase/HCO3--Mediated SOD1 Peroxidase Activity Stimulates DCFH Oxidation to DCF. The addition of Cys to an incubation mixture in phosphate buffer containing SOD1, DCFH, and DTPA and HCO3caused a time-dependent increase in DCF absorbance at 505 nm (Figure 5A). Figure 5B,C shows the timedependent increase in the UV/vis absorbance of DCF as a function of Cys concentration and HCO3-. Figure 5D,E shows the time-dependent increase in DCF fluorescence as a function of Cys and HCO3- concentrations in phosphate buffer containing SOD1, DCFH, and DTPA.

Karunakaran et al.

Figure 5. Oxidation of dichlorodihydrofluorescein by Cys/ HCO3--mediated SOD1 peroxidase activity. (A) Time-dependent optical spectra of DCF obtained from incubations containing bovine SOD1 (15 µM), Cys (500 µM), HCO3- (25 mM), and DCFH (40 µM) in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 µM). The optical maximum at 505 nm indicated by an arrow corresponds to the absorbance of the oxidation product, DCF. (B) Time-dependent formation of DCF absorbance was monitored optically at 505 nm. Incubation conditions are the same as in panel A, except that Cys concentrations were varied. (C) Time-dependent formation of DCF absorbance was monitored optically at 505 nm. Incubation conditions were the same as in panel A, except that HCO3concentrations were varied. (D) Time-dependent formation of DCF fluorescence was monitored using the excitation and emission wavelengths at 495 and 520 nm. Incubation conditions were the same as panel B, except that DCF was detected by fluorescence. (E) Time-dependent formation of DCF fluorescence was monitored as above except at different concentrations of HCO3-.

Characterization of the Nature of Oxidant Formed from the SOD1/HCO3-/Cys System: Measurement of Rate Constants. In the present study, we show that H2O2 generated in situ from the thiol oxidase activity of SOD1 is responsible for stimulating HCO3--dependent SOD1 peroxidase activity. Because of the slow in situ generation of H2O2, it was possible to continuously monitor the initial rates (R) of formation of hydroxylated spin adducts (e.g., DMPO-OH, BMPO-OH). Figure 6A shows the plot of Ro/R vs [inhibitor]/[DMPO] for various inhibitors. The slope of the straight line is the ratio between the second-order rate constants of inhibitor and DMPO with the reactive species (i.e., kinhibitor/kDMPO). The larger the slope, the higher is the rate constant between the oxidant and the inhibitor. The second-order rate constants between various inhibitors and the oxidant formed in the SOD1/Cys/HCO3- system (eq 7) are listed in Table 1. Comparison of these rate constants with the reported values for •OH radical indicates that the oxidant

HCO3- Accelerates SOD1/Thiol Oxidase-Mediated Activity

Figure 6. Effect of inhibitors on the kinetics of DMPO-OH formation. The initial rates of DMPO-OH formation in the absence of inhibitor (Ro) and in the presence of various inhibitors (R) at suitable concentrations where linear rates were observed under similar conditions as in Figure 2. The values of Ro/R are plotted vs [inhibitor]/[DMPO]. The rate constant for the reaction between CO3•- and inhibitors was determined from the slope as defined by k[inhibitor]/k[DMPO]. Table 1. Second-Order Rate Constants between CO3•and •OH and Various Inhibitors k CO3•-/inhibitor (M-1 s-1) inhibitor DMPO Cys tryptophan urate ascorbate histidine glutathione phenylalanine DCFH

k •OH/inhibitor (M-1 s-1) 4.3 × 109 4.7 × 1010 1.3 × 1010 7.2 × 109 5.7 × 109 2.3 × 1010 6.9 × 109

this study 2.7 ( 0.5 × 106 6.9 ( 0.3 × 108 1.3 ( 0.4 × 109 2.7 ( 0.5 × 1010 9.7 ( 0.4 × 106 6.4 ( 0.5 × 106 urate > Trp > His > GSH > DMPO > Phe. Cyclic nitrones (e.g., DMPO) react with CO3•- moderately rapidly (Table 1). However, this rate constant was nearly 1000-fold smaller as compared with • OH trapping with DMPO (Table 1). Ascorbate is the most effective scavenger of CO3•- with a measured rate constant of 2.7 × 1010 M-1 s-1. Uric acid, another well-known antioxidant, reacts fairly rapidly with CO3•- (1.3 × 109 M-1 s-1). DCFH is widely used to measure oxidative stress from products of H2O2 in cells (18). We have determined the rate constant between CO3•- and DCFH to be 1.1 × 109 M-1 s-1 (Table 1). Thus, HCO3- stimulated DCF fluorescence by SOD1 could be attributed to the rapid reaction between CO3•- and DCFH. Enhanced formation of DCF was observed in cells transfected with G93A mutant SOD1 (25, 26). Proposed Mechanism of HCO3-/SOD1-Dependent Thiol Oxidation. A proposed reaction pathway for the accelerated thiol oxidase activity in the presence of HCO3- is described in Scheme 1. Using a kinetic model (Scheme 1), we simulated the loss and regeneration of SOD-Cu2+ in the presence of Cys and bicarbonate anion. The rate constants derived for achieving the best fit between the experimental (open circles in Figure 1F) and the theoretical data (solid line in Figure 1F) are given in Scheme 1. In conclusion, we report in this study that HCO3stimulates the peroxidase activity of SOD1 in the presence of thiols. This peroxidase activity further accelerates thiol loss and oxygen consumption resulting in enhanced thiol oxidase activity via formation of a diffusible CO3•species at the active site that promotes the oxidation of various peroxidatic substrates. Bicarbonate potentially amplifies the pro-oxidant thiol oxidase activity of SOD1 via generation of CO3•-.

Acknowledgment. This work was supported by NIH Grants NS40494 and EB01980. C.K. expresses thanks to the Managing Board, VHNSN College, Virudhunagar, India, for his sabbatical leave. Supporting Information Available: Differential equations constructed from reactions shown in Scheme 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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