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J Biol Chem, Vol. 274, Issue 40, 28233-28239, October 1, 1999

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase
ROLE OF CARBONATE ANION RADICAL^*

Steven P. A. Goss, Ravinder J. Singh, and B. Kalyanaraman

From the Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro--tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H₂O₂-mediated nitration of -tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H₂O₂-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H₂O₂-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H₂O₂, suggest that SOD1/H₂O₂ oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H₂O₂ by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H₂O₂. However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, it was reported that the bicarbonate anion (HCO₃)¹ is required for peroxidase activity and the peroxidase function of copper-zinc superoxide dismutase (SOD1) (1). The peroxidase activity of SOD1 was determined using the electron spin resonance (ESR) technique to monitor the oxidation of spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to the DMPO-hydroxyl radical adduct (DMPO-OH). Additional evidence for enhanced peroxidase activity was obtained by measuring the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) to ABTS radical cation (1). The investigators concluded that the bicarbonate anion, anchored to arginine 141 at the active site of SOD1, facilitated the redox cleavage of H₂O₂ that led to enhanced peroxidase activity (1). In the present study, this interpretation is challenged, and an alternate mechanism for HCO₃-mediated increase in SOD1 peroxidase activity is provided.

Nearly 25 years ago, Hodgson and Fridovich demonstrated that the "copper-bound hydroxyl radical," SOD-Cu²⁺-^·OH, which is generated in the reaction between SOD1 and H₂O₂, oxidizes several anionic ligands including formate, azide, and nitrite anions (2, 3). These small molecular weight anionic ligands are presumed to be oxidized by the oxidant formed at the active site of SOD1. The oxidizing potential of this putative SOD-Cu²⁺-^·OH species is similar to that of the "free" hydroxyl radical, which is considerably higher than those associated with conventional peroxidases (4). The one-electron oxidation potential for the HCO₃/carbonate radical anion (CO₃) couple is +1.59 V (5, 6), which makes oxidation of HCO₃ by SOD-Cu²⁺-^·OH to CO₃ thermodynamically feasible (7, 8). CO₃, although less reactive than the hydroxyl radical, is a more selective oxidant that may diffuse over a longer distance, thereby causing oxidative damage to distant biological targets (9, 10).

We measured the peroxidase activity of SOD1 under anaerobic conditions by using the nitrite anion (NO₂) as a peroxidase substrate (4). The oxidation of NO₂ to the nitrogen dioxide free radical (NO₂^·) was measured by monitoring the formation of -tocopheryl quinone (-TQ) and 5-nitro--tocopherol (NGTH) (4, 11-14). Our results clearly demonstrate that the bicarbonate anion is not required for the peroxidase activity of SOD1; however, bicarbonate enhances oxidation of - and -tocopherols in the presence and absence of the nitrite anion. In addition, bicarbonate provided protection against H₂O₂-dependent inactivation of SOD1. Taken together, these data suggest that SOD1 and H₂O₂ are able to oxidize HCO₃ to CO₃, a more selective oxidant, that leads to amplification of the peroxidase activity of SOD1. Biological implications of the oxidation of HCO₃, a ubiquitous cellular and plasma component, in free radical biology are discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SOD1 (bovine) was purchased from Roche Molecular Biochemicals. 1,2-Dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) was purchased from Avanti Polar Lipids (Alabaster, AL). -Tocopherol (-TH), -tocopherol (-TH), and H₂O₂ were obtained from Sigma.

Synthesis of Oxidation Products-- -TQ was synthesized as previously reported (11, 15). NGTH was synthesized by incubating -TH (2 mM) with ONOO (2 mM) in an acetonitrile/methanol mixture (3:1 v/v) for 10 min. NGTH was extracted with heptane and purified by HPLC.

Liposome Preparation-- Liposomes were synthesized from DLPC (16). Methanolic solutions of -TH, -TH, -TQ, or NGTH were added to the phospholipid, which was then dried down under a stream of nitrogen and placed in a vacuum dessicator overnight. Multilamellar liposomes were prepared by hydration of the dried lipid with phosphate buffer (200 mM, pH 7.4) containing diethylenetriaminepentaacetic acid (DTPA) (100 µM) and thorough mixing. Unilamellar liposomes were prepared from the multilamellar liposomes by freeze-thawing five times using liquid nitrogen and extrusion through a 0.2-µm polycarbonate filter (Nucleopore, Pleasanton, CA) five times using an extrusion apparatus (Lipex Biomembranes, Inc., Vancouver, BC).

Tocopherol Measurement-- Detection and separation of -TH, -TH, and their oxidation products were performed on a HPLC system equipped with a UV detector. Samples (usually 200 µl) were mixed with water (200 µl) and ethanol (400 µl) and vortexed for 60 s. Heptane (400 µl) was added to the samples and vortex-mixed for an additional 60 s. Samples were centrifuged for 10 min at 10,000 rpm to separate the organic and aqueous phases. The organic layer (top) was removed, dried down under a stream of nitrogen, and stored at 20 °C until analysis (<18 h). The samples were dissolved in methanol and analyzed by HPLC. The mobile phase was methanol/water (95:5) for 10 min, graded to 100% methanol over a 5-min period, and followed by 5 min at 100% methanol. The stationary phase was an analytical C₁₈ reversed phase column (Partisil ODS-3, 5-µm particle size, Whatman Inc., Clifton, NJ). UV detection at  = 290 nm was used to quantify -TH, -TH, and NGTH levels, and  = 266 nm was used to quantify -TQ.

Measurement of SOD1 Activity-- SOD1 activity was measured using the ferricytochrome c (cyt c) reduction assay (4, 17). Briefly, xanthine (0.5 mM) and xanthine oxidase (0.05 units/ml) were incubated with cyt c (20 µM) in phosphate buffer (200 mM, pH 7.4). The rate of cyt c reduction by the superoxide anion was measured in the presence and absence of SOD1. To investigate the effect of HCO₃ and NO₂ on SOD1 inactivation by H₂O₂, SOD1 (1 mg/ml) was incubated with H₂O₂ (1 mM) at 37 °C in the presence and absence of HCO₃ and NO₂. SOD1 activity was measured after diluting an aliquot (5 µl) of the reaction mixture with 1 ml of the assay mixture (phosphate buffer containing 1 mM DTPA and 1000 units/ml catalase) (18).

Measurement of Copper and Zinc Release during Inactivation of SOD1 by H₂O₂-- To investigate the effect of HCO₃ on the release of copper and zinc from the enzyme during H₂O₂-mediated SOD1 inactivation, SOD1 (1 mg/ml) was incubated with H₂O₂ (1 mM) in Chelex-treated phosphate buffer (200 mM, pH 7.4) at 37 °C in the presence of 4-pyridylazaresorcinol (PAR, 100 µM). The change in absorbance due to Cu²⁺- and Zn²⁺-PAR complex formation was followed for 3 h at 500 nm. The amount of zinc released during this process was calculated by adding 1.6 mM of nitrilotriacetic acid at the end of the reaction. The decrease in absorbance corresponds to Zn²⁺-nitrilotriacetic acid complex formation (19). The amount of copper released during this process was calculated by the subsequent addition of 0.8 mM EDTA to the incubation mixture as previously reported (19). The molar extinction coefficient of the Cu²⁺- and Zn²⁺-PAR complex remained unchanged up to 250 mM HCO₃.

ESR Measurements-- The formation of -tocopheroxyl (-T^·) and 5-nitro--tocopheroxyl (NGT^·) radicals was monitored by ESR (4, 20). ESR measurements were performed at ambient temperature using a Varian E-109 spectrometer operating at 9.5 GHz (X-band) and 100 kHz field modulation. A typical incubation for ESR experiments consisted of SOD1 (10 mg/ml), H₂O₂ (10 mM); bicarbonate (25 mM); and -TH, -TH, or NGTH (2 mM) incorporated into DLPC liposomes (100 mM) in 0.25 ml of phosphate buffer (200 mM, pH 7.4) containing DTPA (100 µM). The reaction was initiated by the addition of H₂O₂. Samples were immediately analyzed in a quartz flat cell.

Conjugate Diene Measurements-- L--Lecithin liposomes (250 µg/ml) were incubated with SOD1 (0.2 mg/ml) and H₂O₂ (1 mM) in Chelex-treated phosphate buffer (200 mM, pH 7.4) with the transition metal ion chelator DTPA (100 µM) at 37 °C. The changes in absorbance were continuously monitored at 234 nm to detect the formation of conjugated dienes (21).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HPLC Analysis of -TH, -TH, and Their Oxidation and Nitration Products-- -TH and -TH were used as probes to monitor the peroxidase activity of SOD1 in the presence of H₂O₂ (4). In order to limit CO₂ contamination, all solutions were degassed and stored in a nitrogen glove box. After a minimum of 48 h, experiments were performed in the nitrogen glove box. A buffer concentration of 200 mM phosphate was used to prevent changes in pH after the addition of bicarbonate solutions. The pH was determined both before and after the termination of all reactions to ensure that the bicarbonate effect was not due to pH changes.

Fig. 1 shows typical HPLC traces obtained when analyzing large unilamellar DLPC liposomes containing -TH, -TH, NGTH, or -TQ (structures shown in insets). Fluorescence detection (_ex = 275 nm, _em = 320 nm) was used to detect both -TH (B) and -TH (D) with retention times of 13.7 and 15.9 min, respectively. NGTH (A), the nitration product of -TH, was observed to have a retention time of 18.0 min at 290 nm. -TQ (C), the two-electron oxidation product of -TH, was observed to have a retention time of 11.4 min at 266 nm. All peaks were verified using authentic standards.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   HPLC analysis of -TH, -TH, and their oxidation and nitration products. -TH, -TH, NGTH, and -TQ were incorporated into large unilamellar liposomes and analyzed by HPLC. Typical HPLC traces show the result of the retention times of authentic standards (20 µM each) of NGTH (A), -TH (B), -TQ (C), and -TH (D).

The Effect of HCO₃ on SOD1/H₂O₂-mediated Oxidation/Nitration of Tocopherols-- The time course of SOD1/H₂O₂/NO₂-mediated -TH depletion is shown in Fig. 2A. -TH was incubated with SOD1 (1 mg/ml), H₂O₂ (1 mM), and NO₂ (1 mM) in phosphate buffer (200 mM, pH 7.4) containing DTPA (100 µM). Approximately 10 µM -TH was consumed during the first hour of incubation, after which tocopherol depletion occurred at a slower rate (5 µM during the next hour and 6 µM over the next 3 h). This result can be explained in terms of the reaction between -TH and NO₂^· by SOD1/H₂O₂ (4). The addition of HCO₃ to this system markedly enhanced -TH oxidation and -TQ formation. When 3 mM HCO₃ was added to the solution, little change in -TH consumption occurred during the first hour. However, by the fourth hour of incubation, a small increase in consumption was observed. With increasing HCO₃ concentration, a significant increase in -TH consumption (Fig. 2A) and -TQ formation (Fig. 2B) was observed at each time point.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   The effect of HCO3 on SOD1/H2O2-mediated -TH depletion. -TH was incubated with SOD1 (1 mg/ml), H2O2 (1 mM) and NO2 (1 mM) in phosphate buffer (200 mM) containing DTPA (100 µM). There is a concentration-dependent enhancement of -TH depletion (A) and -TQ formation (B) in the presence of HCO3. C shows the effect of the presence of both NO2 and HCO3 when samples containing SOD1 (1 mg/ml) and H2O2 (1 mM) were incubated for 4 h and analyzed by HPLC (n = 3 ± S.D.).

Fig. 2C (control, t = 0 h) shows the effect of the presence or absence of NO₂ and HCO₃ on SOD1/H₂O₂-mediated oxidation of -TH depletion. Approximately 60 µM -TH was incubated with SOD1 (1 mg/ml) in the presence of H₂O₂ (1 mM). After 4 h (control, t = 4 h), -TH levels remained constant. The addition of NO₂ (1 mM) resulted in the consumption of approximately 20 µM -TH, and the formation of 15 µM -TQ, the two-electron oxidation product of -TH. This clearly demonstrates that the peroxidase activity of SOD1/H₂O₂ is not dependent on the presence of HCO₃. However, the presence of both NO₂ and HCO₃ resulted in the consumption of 45 µM -TH, with a further increase in -TQ formation. When HCO₃ (20 mM) alone was added, approximately 20 µM -TH was consumed; however, in this case, -TQ formation was negligible. Further investigation is required to fully characterize the product(s) formed from the oxidation of -TH by the SOD1/H₂O₂/HCO₃ system.

The time course of SOD1/H₂O₂/NO₂-mediated -TH depletion is shown in Fig. 3A. -TH was incubated with SOD1 (1 mg/ml), H₂O₂ (1 mM), and NO₂ (1 mM) in phosphate buffer (200 mM, pH 7.4) containing DTPA (100 µM). Approximately 15 µM -TH was consumed during the first hour of incubation, after which tocopherol depletion occurred at a slower rate (10 µM during the next 3 h). As before (cf. Fig. 2A), the addition of HCO₃ caused a significant increase in -TH consumption at each time point.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   The effect of HCO3 on SOD1/H2O2-mediated -TH depletion. -TH was incubated with SOD1 (1 mg/ml), H2O2 (1 mM), and NO2 (1 mM) in phosphate buffer (200 mM) containing DTPA (100 µM). There is a concentration-dependent enhancement of -TH depletion (A); however, there was little effect on NGTH formation (B) in the presence of HCO3. C, shows the effect of the presence of both NO2 and HCO3 when samples containing SOD1 (1 mg/ml) and H2O2 (1 mM) were incubated for 4 h and analyzed by HPLC (n = 3 ± S.D.).

The effect of HCO₃ on SOD1/H₂O₂/NO₂-mediated NGTH formation is shown in Fig. 3B. -TH was incubated with SOD1 (1 mg/ml), H₂O₂ (1 mM), and NO₂ (1 mM) in phosphate buffer (200 mM, pH 7.4) containing DTPA (100 µM). During the first hour of incubation, approximately 4 µM NGTH was formed. The addition of HCO₃ had little effect on NGTH formation.

Fig. 3C shows the effect of the presence or absence of NO₂ and HCO₃ on -TH depletion and NGTH formation after a 4-h incubation in the presence of SOD1 (1 mg/ml) and H₂O₂ (1 mM). Approximately 5 µM -TH was consumed over a 4-h period, possibly due to NO₂ contamination (as evidenced by a slight increase in the formation of NGTH, control t = 4 h). In the presence of added NO₂ (1 mM), 20 µM -TH was consumed, and 10 µM NGTH was formed. In the presence of HCO₃ (20 mM), 20 µM -TH was also consumed, but NGTH formation was negligible. When both HCO₃ and NO₂ were present, approximately 90% of -TH was consumed, with a concomitant formation of approximately 8 µM NGTH. This paradoxical result (i.e. enhanced -TH depletion and decreased NGTH formation) can be explained if CO₃ also caused the oxidation of NGTH.

These reactions suggest that HCO₃ is not required for SOD1/H₂O₂/NO₂-mediated nitration of -TH. The presence of HCO₃, however, enhanced the oxidation of -TH and possibly NGTH.

ESR Detection of Tocopheroxyl Radicals: Enhancement by HCO₃-- To probe the involvement of tocopheroxyl radical during HCO₃-enhanced oxidation of tocopherols, we used direct ESR. The addition of H₂O₂ (10 mM) to an incubation mixture containing -TH (2 mM) and SOD1 (10 mg/ml) produced a seven-line ESR spectrum (Fig. 4A, a) characteristic of the -T^· radical. In the presence of HCO₃, there was a marked increase in signal intensity (Fig. 4A, b). Due to a decreased signal-to-noise ratio, a high modulation amplitude was used that restricted our ability to resolve couplings from the methyl protons at carbon-8 and methylene protons at carbon-4. The computer simulation of the -T^· radical is shown in Fig. 4A, c (a_CH3⁵ (3H) = 5.66 G; a_CH3⁷ (3H) = 4.49 G) (20, 22, 23). When either SOD1 or H₂O₂ was excluded, there was no detectable ESR signal (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   HCO3-enhanced formation of -T· and NGT· radicals during the oxidation of -TH and NGTH by SOD1/H2O2. DLPC liposomes containing -TH or NGTH (3.5 mM each) were incubated in the presence of SOD1 (10 mg/ml) and H2O2 (10 mM) in phosphate buffer (200 mM, pH 7.4) at ambient temperature. A, a, ESR spectrum of -T· generated in the presence of SOD1 and H2O2; b, ESR spectrum generated in the presence of 25 mM bicarbonate; c, simulation of the spectrum using the spectral parameters for -T· as described under "Results." B, spectra a-c correspond to the conditions used in panel A, a-c, using NGTH in place of -TH. Spectrometer conditions were as follows: time constant, 0.25 s; microwave power, 40 milliwatts; scan range, 100 G; scan time, 1 min; modulation amplitude, 0.5 G. DLPC liposomes containing NGTH (50 µM) were incubated in the presence of SOD1 (1 mg/ml), H2O2 (1 mM) in the absence (Ca) and presence (Cb) of HCO3 (25 mM). D, traces a and b correspond to the conditions used in panel C, a and b, using NGTH in place of -TH.

Incubation of DLPC liposomes containing NGTH with SOD1/H₂O₂ produced a four-line spectrum (Fig. 4B, a) with a 1:3:3:1 intensity ratio, typical for an electron interacting with three equivalent protons. The addition of 25 mM HCO₃ resulted in the enhancement of this signal (Fig. 4B, b). The computer simulation of this spectrum (dotted lines) is shown in Fig. 4B, c (a_CH3⁷ (3H) = 4.35 G). At this high modulation, the nitrogen coupling could not be resolved.

DLPC liposomes containing -TH (60 µM) were incubated with SOD1 (1 mg/ml) and H₂O₂ (1 mM). Fig. 4C shows HPLC traces of samples incubated in the absence (Fig. 4C, a, 58 ± 1 µM -TH) or presence (Fig. 4C, b, 39 ± 2 µM -TH) of HCO₃ (25 mM) after a 4-h incubation at 37 °C. Incubation of DLPC liposomes containing NGTH (25 µM) with SOD1 (1 mg/ml) and H₂O₂ (1 mM) at 37 °C for 4 h resulted in a reduction in NGTH concentration to 20 ± 1 µM in the absence of HCO₃ (Fig. 4D, a) and to 14 ± 1 µM in the presence of HCO₃ (25 mM, Fig. 4D, b).

The ESR and HPLC results clearly demonstrate that the HCO₃ anion enhanced SOD1/H₂O₂-mediated oxidation of tocopherols via a radical-mediated pathway. It is likely that an oxidant, possibly the CO₃ radical derived from the SOD1/H₂O₂-catalyzed oxidation of HCO₃ anion, is responsible for the increased oxidation of tocopherols.

Lipid Peroxidation by SOD1/H₂O₂/HCO₃-- SOD1/H₂O₂/HCO₃- and SOD1/H₂O₂/NO₂-mediated lipid peroxidation were monitored by measuring conjugated diene formation at 234 nm in L--lecithin liposomes containing DTPA (100 µM). Conjugated diene formation was minimal during the incubation of liposomes in the presence of SOD1 (200 µg/ml) and H₂O₂ (1 mM) in the absence of either HCO₃ or NO₂ (Fig. 5). The addition of NO₂ (1 mM) resulted in an increase in absorbance at 234 nm, corresponding to the formation of conjugated dienes as a function of time. HCO₃ (25 mM) significantly enhanced conjugated diene formation in the presence of NO₂.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of bicarbonate on SOD1/H2O2-mediated lipid peroxidation. L--Lecithin liposomes (250 µg/ml) were incubated in the presence of DTPA (100 µM) with SOD1 (200 µg/ml) and H2O2 (1 mM) at 37 °C in Chelex-treated phosphate buffer (200 mM, pH 7.4) and monitored at 234 nm for conjugated diene formation. The effect of HCO3 (25 mM), NO2 (1 mM) and HCO3 with NO2 on conjugated diene formation was observed. Each trace is the average of triplicate replications.

Protection of H₂O₂-mediated Inactivation of SOD1 by Bicarbonate-- Fig. 6A (inset) shows the rate of superoxide-dependent cyt c reduction, monitored as an increase in absorbance at 550 nm (a). In the presence of SOD1, the increase in absorbance was inhibited (f). After incubation of SOD1 with H₂O₂ for 4 h, SOD1-mediated inhibition of cyt c reduction was less effective (b). The presence of HCO₃ (c), NO₂ (d), or HCO₃ and NO₂ (e) prevented the inactivation of SOD1 by H₂O₂. SOD1 activity was calculated from the initial rate of cyt c reduction and is shown as a percentage of the rates of cyt c observed in the absence (a) or in the presence (f) of SOD1 (n = 3 ± S.D.).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Bicarbonate inhibits H2O2-mediated SOD1 inactivation. A, SOD activity was assayed by monitoring the inhibition of superoxide-dependent cyt c (20 µM) reduction during the reaction of xanthine (500 µM) and xanthine oxidase (0.05 unit/ml) at 37 °C in 200 mM phosphate buffer containing 1000 units/ml catalase and DTPA (100 µM). Enzyme activity is reported as a percentage of the inhibition of cyt c reduction observed in the absence (a, 0%) or in the presence of SOD1 (f, 100%); effect of 1 mM H2O2-inactivated SOD (b), H2O2-treated SOD in the presence of 25 mM HCO3 (c), or H2O2-treated SOD in the presence of 1 mM NO2 (d) or the presence of 25 mM HCO3 and 1 mM NO2 (e) (n = 3 ± S.D.). Inset, the actual UV absorbance traces at 550 nm showing rates of cyt c reduction, corresponding to the conditions described above. B, copper release was observed during enzyme inactivation, as determined by the PAR assay. The presence of HCO3 (25 mM) and NO2 (1 mM) inhibited the release of copper from the enzyme. Traces are representative of triplicate replications.

Inactivation of SOD1 was accompanied by the release of copper and zinc from the active site as determined by the PAR assay. NO₂ (1 mM) and/or HCO₃ (20 mM) inhibited the release of copper and zinc during H₂O₂-mediated inactivation of SOD1 (Fig. 6B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bicarbonate Anion as a Peroxidase Substrate-- The oxidative inactivation of SOD1 caused by H₂O₂ has been attributed to the "peroxidase activity" (2, 3). This "peroxidase activity" results in the generation of a highly potent, "site-specific" hydroxyl radical-like species that inactivates the enzyme by oxidative attack on a histidine residue (His-61) at the active site (24-27) (Scheme 1).

View larger version (12K): [in this window] [in a new window]   Scheme 1.

The putative oxidant, SOD-Cu²⁺-^·OH, reacts with several electron donor compounds to form the corresponding radical while protecting the enzyme from oxidative inactivation. HCO₃ partially protects against H₂O₂-induced inactivation of SOD1 and inhibits copper release. This may be attributed to the oxidation of HCO₃ to CO₃ by the enzyme-bound oxidant (2-4). Unlike the copper-bound hydroxyl radical at the active site, CO₃ is a freely diffusible oxidant that can oxidize target molecules at a distance (9, 10). We attribute the increased peroxidase activity of SOD1 (observed in the presence of HCO₃) to the oxidative reactions catalyzed by CO₃.

Oxidative Reactions of CO₃-- In contrast to CO₂, a reducing radical formed from oxidation of the formate anion, CO₃ is an oxidizing radical (28, 29). For example, CO₂ reacts with O₂ to form O₂, whereas CO₃ does not react with O₂. While CO₂ reduces ^·NO to NO, CO₃ oxidizes ^·NO to NO₂ (30). CO₃ forms a conjugate acid and base form, and this radical is mostly unprotonated and exists as an anion at physiological pH levels (31, 32). The HCO₃-induced depletion of tocopherols in the presence of SOD1 and H₂O₂ reported in the present study can be explained based on the following reactions. HCO₃ enhanced NO₂-dependent oxidation of tocopherols and unsaturated fatty acid in the presence of SOD1 and H₂O₂. We attribute this to a fast electron transfer reaction between CO₃ and NO₂ that results in the formation of NO₂^· (33). NO₂^· can abstract the phenolic hydrogen atom from the tocopherols to form the tocopheroxyl radical (34). NO₂^· reacts with -T^· to form the corresponding quinone -TQ. Alternatively, NO₂^· reacts with the linoleate-type fatty acid (k = 10⁵ to 10⁶ M¹ s¹) to form the lipid-conjugated diene.

CO₃ can participate in electron transfer reactions, addition, or hydrogen abstraction reactions (9, 10). CO₃ reacts rapidly with tyrosine (Tyr-OH), tryptophan, and other phenolic compounds (k = 10⁷ to 10⁸ M¹ s¹) to form the corresponding phenoxyl radicals (35). CO₃ directly reacts at the sulfur site of many sulfur-containing compounds such as glutathione (k  5 × 10⁶ M¹ s¹) and cysteine (k  5 × 10⁷ M¹ s¹) (35).

Reactions similar to those discussed above may explain the HCO₃-induced increase in formation of DMPO-OH and ABTS radical cation (ABTS) observed in the SOD1/H₂O₂ system (1). The DMPO cation radical has been postulated as an intermediate (7, 36). The DMPO will undergo hydrolysis to form DMPO-OH. This proposal is in agreement with our previous spin trapping investigation in oxygen-17-enriched water and H₂O₂ (36). To determine the source of the oxygen atom in the DMPO-OH adduct, we used oxygen-17-enriched hydrogen peroxide ([¹⁷O]-H₂O₂) and water ([¹⁷O]-H₂O). The reaction of SOD1 with [¹⁷O]-H₂O₂ in HCO₃/CO₂ buffer yielded 63% DMPO-¹⁷OH and 37% DMPO-¹⁶OH. In contrast, the relative concentrations of DMPO-¹⁷OH and DMPO-¹⁶OH formed in the Fenton reaction were 90 and 10%, respectively. Since the commercial [¹⁷O]H₂O₂ contained 89% ¹⁷O-labeled H₂O₂, nearly all of the DMPO-OH formed in the Fenton reaction originated from H₂O₂.² In contrast, nearly 35% of DMPO-OH arises from the incorporation of oxygen from water in the SOD1/H₂O₂ reaction. These results were further confirmed using a different spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (36). These results do not support the model previously proposed for HCO₃-assisted hydroxylation of DMPO at the active site of SOD1 (1). Based on that model, one would have expected 100% incorporation of H₂O₂-derived oxygen into DMPO-OH.

Detection of CO₃ at Physiological pH-- The CO₂ radical formed during SOD1/H₂O₂-catalyzed oxidation of the formate anion has been spin-trapped with DMPO to give a DMPO- CO₂ adduct, which gave a characteristic ESR spectrum (28). In contrast, the addition of SOD1 to an incubation mixture containing DMPO, H₂O₂, HCO₃, and DTPA enhanced DMPO-OH formation (1, 36). The DMPO-CO₃ adduct was not stable enough to be detected at physiological pH levels. Recently, using a rapid mixing continuous flow ESR, CO₃ production from peroxynitrite and carbon dioxide was detected at physiological pH levels (32, 37). It is conceivable that CO₃ formed from SOD1/H₂O₂/HCO₃ may be detected using similar experimental techniques.

Biological Implications-- Hodgson and Fridovich (38) reported that the addition of carbonate anion to an aerobic xanthine/xanthine oxidase system produced luminescence. This effect was attributed to a dimerization of CO₃ radical anion (39) formed from the reaction between ^·OH and CO₃² (33) as follows. In the pioneering studies of the peroxidase activity of SOD1, some experiments were performed in carbonate buffer (2, 3). It is likely that H₂O₂-induced inactivation of SOD1 was partially protected by the carbonate anion.

Bicarbonate has been shown to alter the reactivity of reactive nitrogen species (40-48). Peroxynitrite, a potent oxidant formed from a diffusion-controlled reaction between O₂ and ^·NO, has been proposed to react with CO₂ to generate an intermediate that consists of a caged radical pair, CO₃/NO₂^·. The chemiluminescence detected in the reaction between ONOO and CO₂ was attributed to the formation of a carbonate anion radical (49). The CO₂-peroxynitrite reaction facilitated the nitration and oxidation of tyrosine (39). This scenario is similar to the bicarbonate-enhanced reactivity of SOD1/H₂O₂/NO₂ reported in the present study.

HCO₃ is abundant in biological systems. The plasma concentration of HCO₃ is approximately 25 mM (50). Recently, an extracellular SOD1 has been identified in atherogenesis (51). The peroxidase activity of this extracellular SOD1 has been suggested to play an important role in the oxidative modification of lipid. This activity may be amplified in the presence of HCO₃. There is emerging evidence that links familial amyotrophic lateral sclerosis to mutations in the gene encoding cytosolic SOD1 (52). The mutations in SOD1 induce a gain in function that is associated with increased peroxidase activity (53). HCO₃ may influence the radical reactions catalyzed by these mutations of SOD1. Regulation of oxidative reactions by HCO₃ in other physiologically relevant inflammatory processes (e.g. myeloperoxidase) may also be important (54, 55).

In conclusion, in contrast to a previous report (1), evidence presented in this study strongly suggests that HCO₃ is not an absolute requirement for eliciting the peroxidase activity of SOD1. However, HCO₃ enhances the peroxidase activity of SOD1 by acting as a "sacrificial" electron donor and in so doing forms a CO₃ radical intermediate, a selective yet potent biological oxidant. The involvement of CO₃ in biological oxidation seems more ubiquitous (56) and should be taken into consideration in future studies.

	FOOTNOTES

^* This work has been supported by National Institutes of Heath Grants RR01008, HL47250, and HL63119 and by a grant from the Amyotrophic Lateral Sclerosis Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Biophysics Research Institute, Medical College of Wisconsin, 8701 Watertown Plank Rd., P.O. Box 26509, Milwaukee, WI 53226. Tel.: 414-456-4035; Fax: 414-456-6512; E-mail: balarama@mcw.edu.

² There is no ¹⁷O exchange between DMPO-¹⁶OH and H₂¹⁷O or H₂¹⁷O₂ (36). The commercially available [¹⁷O]H₂O₂ has been kept in [¹⁶O]H₂O without evidence of exchange for years.

	ABBREVIATIONS

The abbreviations used are: HCO₃, bicarbonate anion; -TH, -tocopherol; -T^·, -tocopheroxyl radical; -TQ, -tocopheryl quinone; CO₃, carbonate radical anion; DTPA, diethylenetriaminepentaacetic acid; DLPC, 1,1-dilauroyl-sn-glycero-3-phosphatidylcholine; DMPO, 5-5'-dimethyl-1-pyrroline N-oxide; DMPO-OH, 5-5'-dimethyl-1-pyrroline N-oxide-hydroxyl radical adduct; ESR, electron spin resonance; -TH, gamma-tocopherol; NO₂, nitrite anion; NO₂^·, nitrogen dioxide free radical; NGTH, 5-nitro-gamma tocopherol; NGT^·, 5-nitro-gamma-tocopheroxyl radical; ONOO, peroxynitrite and peroxynitrous acid; SOD1, copper-zinc superoxide dismutase; SOD-Cu²⁺-^·OH, copper-zinc superoxide dismutase with "bound" hydroxyl radical; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid; HPLC, high pressure liquid chromatography; cyt c, cytochrome c; PAR, 4-pyridylazaresorcinol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES