Rate Constants for the Reaction of the Carbonate Radical with Compounds of Biochemical Interest in Neutral Aqueous Solution

110

115

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1 WT In a pair of publications, Hodgson and Fridovich demonstrated that bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD or SOD1) 1 exhibits a nonspecific peroxidase activity (1, 2). They provided evidence for formation of a potent oxidant in the presence of H 2 O 2 that was ascribed to a copper-bound hydroxyl radical, at the active site of bovine SOD1. Although the rate constant for the reaction between SOD1 and H 2 O 2 is very low (3), it was shown that the bovine SOD1 peroxidase activity could oxidize a variety of small molecular weight anionic ligands (azide, nitrite, formate, etc.) that are accessible to the active site (1, 2, 4, 5). However, a perplexing aspect of this peroxidase activity was that even larger molecules (e.g. 2,2Ј-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid, urate) that are not accessible to the active site of SOD1 were also oxidized (6). It was later discovered that the bicarbonate anion (HCO 3 Ϫ ) that is present in the buffer was responsible for Cu,Zn-SOD peroxidase-dependent oxidations of substrates in the bulk solution (7,8). It was proposed that the copper-bound oxidant (Cu 2ϩ -OH or Cu III O) could oxidize the HCO 3 Ϫ anion (a physiologically relevant molecule) to the carbonate anion radical (CO 3 . ), a potent oxidant that diffuses out of the active site and causes substrate oxidation (7, 8). The fact that CO 3 . , and not hydroxyl radical, is the primary oxidant produced by SOD1-mediated peroxidase activity is physiologically significant, because CO 3 . has a much longer half-life (than hydroxyl radical) and can, therefore, diffuse away from the active site and oxidatively modify critical cellular targets. Liochev et al. (7) suggested that the nitrone spin trap, 5,5Ј-dimethyl-1-pyrroline-N-oxide (DMPO), is oxidized and hydrolyzed by CO 3 . . Zhang et al. (6) provided the experimental proof for this hypothesis. Using electron spin resonance (ESR) spin trapping in the presence of oxygen-17-labeled water, the investigators showed that the oxygen atom in the DMPO-OH adduct is derived totally from water. Zhang et al. (6) also reported that HCO 3 Ϫ -dependent peroxidase activity can be measured using a variety of methods including fluorescence and optical spectroscopy. More recently, Hink et al. (9) demonstrated that an extracellular SOD (SOD3 or ecSOD) enhanced the hydroxylation of a cyclic nitrone spin trap in the presence of H 2 O 2 and HCO 3 Ϫ . In this work, we report that human Cu,Zn-superoxide dismutase (hSOD1 WT ) also exhibits a HCO 3 Ϫ -dependent peroxi-* This work was supported by National Institutes of Health Grants RR01008, NS40494, and NS40819 (to J. P. C.) and the ALS Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. . , carbonate radical anion; DMPO, 5,5Ј-dimethyl-1-pyrroline-N...

Section: Inhibition Of Hsod1 Aggregation By Nitrone Spin Traps-supporting

confidence: 89%

Bicarbonate-dependent Peroxidase Activity of Human Cu,Zn-Superoxide Dismutase Induces Covalent Aggregation of Protein

Zhang

Andrekopoulos

Joseph

et al. 2003

110

115

“…Consequently, the higher RSO ⅐ concentrations detected in the presence of HCO 3 Ϫ /CO 2 at pH 7.4 than at pH 5.4 (Figs. 3, B and D, and 4, B and D) are likely to be due to the usually higher rate constants of thiol oxidation at alkaline pH values (25,36,49). Detection of RSO ⅐ was possible in incubations containing equimolar concentrations of peroxynitrite and RSH (Figs.…”

Section: Fig 2 Effects Of Hco 3 ؊ /Co 2 On Oxygen Consumption Durinmentioning

confidence: 99%

Carbon Dioxide Stimulates the Production of Thiyl, Sulfinyl, and Disulfide Radical Anion from Thiol Oxidation by Peroxynitrite

Bonini

Augusto

2001

173

105

Reaction of peroxynitrite with the biological ubiquitous CO 2 produces about 35% yields of two relatively strong one-electron oxidants, CO 3 . and ⅐ NO 2 , but the remaining of peroxynitrite is isomerized to the innocuous nitrate. Partial oxidant deactivation may confound interpretation of the effects of HCO 3 ؊ /CO 2 on the oxidation of targets that react with peroxynitrite by both one-and two-electron mechanisms. Thiols are example of such targets, and previous studies have reported that HCO 3 ؊ / CO 2 partially inhibits GSH oxidation by peroxynitrite at pH 7.4. To differentiate the effects of HCO 3 ؊ /CO 2 on twoand one-electron thiol oxidation, we monitored GSH, cysteine, and albumin oxidation by peroxynitrite at pH 5.4 and 7.4 by thiol disappearance, oxygen consumption, fast flow EPR, and EPR spin trapping. Our results demonstrate that HCO 3 ؊ /CO 2 diverts thiol oxidation by peroxynitrite from two-to one-electron mechanisms particularly at neutral pH. At acid pH values, thiol oxidation to free radicals predominates even in the absence of HCO 3 ؊ /CO 2 . In addition to the previously characterized thiyl radicals (RS ⅐ ), we also characterized radicals derived from them such as the corresponding sulfinyl (RSO ⅐ ) and disulfide anion radical (RSSR ⅐ ؊ ) of both GSH and cysteine. Thiyl, RSO ⅐ and RSSR ⅐ ؊ are reactive radicals that may contribute to the biodamaging and bioregulatory actions of peroxynitrite.Peroxynitrite 1 (ONOO Ϫ ϩ ONOOH), which is formed by the fast reaction between ⅐ NO and O 2 . , has been receiving increasing attention as a mediator of human diseases and as a toxin against invading microorganisms (1-5). The compound is a potent oxidant that is able to oxidize and nitrate a variety of biological targets by mechanisms that are presently being elucidated (6 -16). In general terms, peroxynitrite-mediated oxidations are either bimolecular, first order on peroxynitrite and biomolecule concentration, or unimolecular, first order on peroxynitrite and independent of biomolecule concentration. Bimolecular processes can result in product yield either around stoichiometry and over, as is the case for thiol oxidation (17), or around 35% , as is the case for carbon dioxide (CO 2 ) oxidation (10, 14 -16). Presently, most investigators accept that product yields around 30% are characteristic of peroxynitrite-mediated free radical processes. Indeed, it has been established that ONOO Ϫ protonation (pK a ϭ 6.6) leads to its fast decomposition (k ϭ 0.17 s Ϫ1 at pH 7.4) to yield approximately 70% nitrate and 30% hydroxyl radical ( ⅐ OH) and nitrogen dioxide ( ⅐ NO 2 ) (Fig. 1, path 1) (7)(8)(9)(11)(12)(13)(14). These radicals are the species responsible for peroxynitrite-mediated unimolecular oxidations. In the presence of HCO 3 Ϫ , ONOO Ϫ decomposes much faster because of its reaction with CO 2 (k ϭ 2.6 ϫ 10 4 M Ϫ1 s Ϫ1 at pH 7.4, 25°C) (18, 19) to produce approximately 65% nitrate and 35% carbonate radical anion (CO 3 . ) and ⅐ NO 2 (Fig. 1, path 2) (10, 14 -16).The fast rate constant of this reaction and...

“…CO 3 . reacts rapidly with tyrosine (Tyr-OH), tryptophan, and other phenolic compounds (k ϭ 10 7 to 10 8 M Ϫ1 s Ϫ1 ) to form the corresponding phenoxyl radicals (35).…”

Section: Oxidative Reactions Of Comentioning

confidence: 99%

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

Goss

Singh

Kalyanaraman

1999

128