Bicarbonate Is Required for the Peroxidase Function of Cu,Zn-Superoxide Dismutase at Physiological pH

Sankarapandi, Sornampillai; Zweíer, Jay L.

doi:10.1074/jbc.274.3.1226

Cited by 86 publications

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“…The investigators concluded that the bicarbonate anion, anchored to arginine 141 at the active site of SOD1, facilitated the redox cleavage of H 2 O 2 that led to enhanced peroxidase activity (1). In the present study, this interpretation is challenged, and an alternate mechanism for HCO 3 Ϫ -mediated increase in SOD1 peroxidase activity is provided.…”

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confidence: 57%

“…Recently, it was reported that the bicarbonate anion (HCO 3 Ϫ ) 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 5,5Ј-dimethyl-1-pyrroline-N-oxide (DMPO) to the DMPO-hydroxyl radical adduct (DMPO-OH).…”

mentioning

confidence: 99%

“…ϩ 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 H 2 O 2 that led to enhanced peroxidase activity (1).…”

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confidence: 99%

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Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase

Goss

Singh

Kalyanaraman

1999

Journal of Biological Chemistry

130

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confidence: 57%

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confidence: 99%

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Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase

Goss

Singh

Kalyanaraman

1999

Journal of Biological Chemistry

130

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“…While considering carbonic anhydrases, it is reasonable to ask whether this ubiquitous family of enzymes might play a defensive role by catalyzing the hydration of CO 2 , which can be oxidized to CO 3 •Ϫ , in contrast to HCO 3 Ϫ , which cannot. In support of this supposition is the report by Götz et al (10) (4) and that HCO 3 Ϫ specifically binds in the solvent access channel of SOD1 and, while so bound, becomes oxidized, at which time the resulting CO 3 •Ϫ protrudes sufficiently to oxidize substrates in the bulk solution (6). Reaction conditions shown are essentially as in Fig.…”

Section: Discussionmentioning

confidence: 87%

CO ₂ , not \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation}{\mathrm{HCO}}_{3}^{-}\end{equation}\end{document}, facilitates oxidations by Cu,Zn superoxide dismutase plus H ₂ O ₂

Liochev

Fridovich

2004

Proc. Natl. Acad. Sci. U.S.A.

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The Cu,Zn superoxide dismutase catalyzes HCO 3 ؊ -dependent oxidations by H 2O2. This activity has been shown to depend on the creation of a bound oxidant at the Cu(II) by interactions with H 2O2. The bound oxidant was then thought to oxidize HCO 3 ؊ to CO 3 •؊ , which diffuses into the bulk solution and there oxidizes diverse substrates. We now find that CO 2 rather than HCO 3 ؊ facilitates the peroxidations catalyzed by Cu,Zn superoxide dismutase. This fact was shown by a lag in the rate of peroxidation of NADPH when NaHCO 3 ؊ was added last and by a burst in the rate when aqueous CO 2 was added last. Both the lag and the burst were eliminated by carbonic anhydrase.Ϫ dramatically increases the rate of oxidation of diverse substrates by Cu,Zn superoxide dismutase (SOD1) plus H 2 O 2 , while only modestly increasing the intrinsically slow inactivation of this enzyme by H 2 O 2 (1-4). The following reactions have been advanced as part of a scheme to explain the behavior of this system (1).Thus, the cuprous enzyme produced in reaction i is oxidized in reaction ii by a second H 2 O 2 to yield a strong bound oxidant, which can be written as Cu(I)O, Cu(II)OH, or Cu(III). This bound oxidant can either oxidize an adjacent histidine residue and in so doing inactivate the enzyme, or it can oxidize HCO 3 Ϫ to the carbonate radical (CO 3 •Ϫ ) that is free to diffuse into the bulk solution and cause further oxidations. Before leaving the active site, the carbonate radical can oxidize residues in the ligand field of the copper and thus cause inactivation.HCO 3 Ϫ was assumed to be the species in the carbonate buffer that facilitated the oxidations observed. This was the case because, at neutral to mildly alkaline pH, it is the major species. It also seemed reasonable that an active site evolved to deal with the monovalent anion O 2 Ϫ would preferentially react with other monovalent anions. Nevertheless, the experiments described below establish that at pH 7.4, it is CO 2 rather than HCO 3 Ϫ that profoundly facilitates the oxidation of NADPH by SOD1 plus H 2 O 2 . Materials and MethodsSOD1 was from Chemie Grünenthal (Aachen, Germany); H 2 O 2 , from Mallinckrodt; and NaHCO 3 , from Fisher; whereas NADPH and carbonic anhydrase were from Sigma. NADPH at 0.1 mM was reacted with 0.1 or 0.2 mg͞ml SOD1 and 10 mM H 2 O 2 in 100 mM potassium phosphate at pH 7.4. When HCO 3 Ϫ was used to facilitate the oxidation of NADPH (followed at 340 nm), it was added to 20 mM from a 1.0 M stock of NaHCO 3 in water. When CO 2 was used, it was taken from ice-cold water saturated with CO 2 gas at 1.0 atmosphere (atm) (1 atm ϭ 101.3 kPa) that would contain 76 mM CO 2 . Carbonic anhydrase when used was present at 0.1 mg͞ml. Additional experimental details are presented in the legends to Figs. 1 and 2. ResultsLine 1 in Fig. 1 demonstrates an immediately linear rate of NADPH oxidation that was seen when the HCO 3 -dependent oxidation by SOD1 plus H 2 O 2 reaction was started by adding either SOD1 or H 2 O 2 . However, when NaHCO 3 was the last component ...

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“…Any valid mechanism must explain the dual requirement for HCO 3 Ϫ and CO 2 and also the involvement of O 2 Ϫ . Moreover, analogy with the Cu,ZnSOD ϩ H 2 O 2 system indicates that CO 3 •Ϫ is a likely participant, whereas HO • is not (1,(3)(4)(5)(6)(7)(8) Ϫ , yet rapid H 2 O 2 consumption is a hallmark of this process.…”

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confidence: 99%

Carbon dioxide mediates Mn(II)-catalyzed decomposition of hydrogen peroxide and peroxidation reactions

Liochev

Fridovich²

2004

Proc. Natl. Acad. Sci. U.S.A.

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Mn(II) can catalyze the decomposition of H2O2 and, in the presence of H 2O2, can catalyze the oxidation of NADH. Strikingly, these processes depend on the simultaneous presence of both CO 2 and HCO 3 ؊ . This explains the exponential dependence of the rates on Materials and MethodsNaHCO 3 , MnCl 2 , sodium pyrophosphate, and H 2 O 2 were from Mallinckrodt; Tris was from Merck; catalase was from Roche Molecular Biochemicals; human MnSOD was from Biotechnology General (Rehovot, Israel); and NADH was from Sigma. Reaction mixtures usually contained 10 mM H 2 O 2 , 0.1 mM MnCl 2 , and 20 mM NaHCO 3 in 100 mM Tris buffer at pH 7.4 and room temperature (23°C). Reactions were followed spectrophotometrically, and in cases where the addition of reactants entailed sudden changes in absorbance, due either to dilution or the absorbance of the reagent added, such changes were corrected in the data shown. When absorbance changes with time were too rapid to record, they are shown as dashed lines as in Fig. 3. CO 2 was added as ice-cold water saturated with CO 2 gas. ResultsAbsorption Spectra. Addition of the third component (H 2 O 2 or MnCl 2 , or NaHCO 3 ), to Tris-buffered solutions of the other two, initiated the changes in absorption spectrum shown in Fig. 1. It should be noted that several buffering species were explored, including phosphate, cacodylate, and Hepes, but only Tris supported the changes observed. Fig. 1 A shows that the most rapid (0 -3 min) was an increase in absorbance in the range of 250 -350 nm and a decrease at wavelengths Ͻ250 nm. At longer reaction times (0 -20 min), absorbance appeared at Ϸ270 nm, whereas the absorbance Ͻ250 nm first decreased and then increased, as shown in Fig. 1B. At still longer times (Fig. 1C), the band at Ϸ270 nm became more pronounced, a shoulder developed at Ϸ300 nm, and absorbance Ͻ250 nm decreased.The species responsible for the absorbance centered at 270 nm is some stable product of Tris oxidation, as similar absorbance is seen in Tris buffer that has aged for many months. The kinetics of the absorbance change at 270 nm is shown in Fig. 2. Starting the reaction by adding NaHCO 3 caused a small but rapid increase at 270 nm, which was followed by a slower increase that was not interrupted by late addition of 48 g͞ml MnSOD, 0.5 mM pyrophosphate, or 120 units͞ml catalase, any of which inhibited if present at the outset. The initial rapid increase at 270 nm was due to the rapidly formed species absorbing in the 250-to 350-nm region. Evidently, the stable A 270 species is produced from a preformed precursor and does not depend on the continued presence of H 2 O 2 , O 2 Ϫ , or Mn(III). The latter deduction is based on the abilities of catalase to remove H 2 O 2 , MnSOD to remove O 2 Ϫ , and pyrophosphate to complex and thus trap Mn(III).The time course of the broad band followed at Ϸ300 nm is illustrated in Fig. 3. Addition of NaHCO 3 to the buffered solution of Mn(II) ϩ H 2 O 2 caused a rapid increase in absorbance that approached a plateau. This plateau evidently represented a...

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Bicarbonate Is Required for the Peroxidase Function of Cu,Zn-Superoxide Dismutase at Physiological pH

Cited by 86 publications

References 50 publications

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase

Carbon dioxide mediates Mn(II)-catalyzed decomposition of hydrogen peroxide and peroxidation reactions

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