Original contributionOxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite
Introduction
Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radical are potent oxidants generated in aerobic organisms during the normal cellular metabolism and in response to external factors. Increased levels of ROS and their toxic by-products can have serious effects on membrane lipids, nucleic acids, and proteins. To protect against the toxicity of ROS and also to maintain the normal redox potential in the cell, aerobic organisms have developed complex antioxidant mechanisms involving both nonenzymatic (ascorbic acid, carotenes, α-tocopherol, glutathione, ubiquinones, etc.) and enzymatic (superoxide dismutase, catalase, glutathione peroxidase, etc.) systems. A new addition to the catalog of antioxidant enzymes is a large family of novel peroxidases, designated peroxiredoxins, which has been discovered in recent years [1], [2], [3], [4]. Peroxiredoxins are distributed widely in nature from bacteria to man and found abundantly in various tissues and cells [2], [3], [4]. All peroxiredoxins so far characterized contain a highly conserved redox-active cysteine, which is located near the amino terminus (designated Cys N) and is oxidized by peroxides. Many peroxiredoxins also contain a second conserved cysteine located in a region closer to the carboxyl terminus (designated Cys C) of the protein, and therefore are referred to as 2-Cys peroxiredoxins, while peroxiredoxins, which lack the second conserved cysteine, are referred to as 1-Cys peroxiredoxins [1], [2]. The mammalian genome encodes six distinct peroxiredoxins [5], which can be classified into three subgroups based on their amino acid sequences and catalytic mechanisms. The first subgroup comprises four distinct 2-Cys peroxiredoxins that reduce peroxides using thioredoxin as an electron donor. The model of the catalytic mechanism of mammalian 2-Cys peroxiredoxins was built mainly on the basis of studies of bacterial and yeast 2-Cys peroxiredoxins [6], [7], [8], [9], [10], [11], [12]. According to this model, peroxides oxidize Cys N of one subunit to an intermediate (cysteine sulfenic acid), which then reacts with Cys C of another subunit to produce an intermolecular disulfide. The disulfide is subsequently reduced by thioredoxin. The second subgroup comprises human, mouse, and rat peroxiredoxins designated PrxV [5], [13], [14], [15]. Prx V resembles 1-Cys peroxiredoxin in that it does not contain Cys C. But unlike 1-Cys peroxiredoxin, Prx V forms an intramolecular disulfide as a reaction intermediate; the transiently formed sulfenic acid of Cys N reacts with the SH-group of one of the cysteines present in the molecule. Thus, the intramolecular disulfide intermediate of Prx V is distinct from the intermolecular disulfide intermediate of 2-Cys peroxiredoxins. Thioredoxin was found to be able to support the peroxidase activity of human PrxV [13]. The third subgroup consists of 1-Cys peroxiredoxins, which, unlike 2-Cys peroxiredoxins and Prx V, form neither an intermolecular nor intramolecular disulfide intermediate [16], [17]. A recent study on the crystal structure of human recombinant 1-Cys peroxiredoxin has shown that the intermediate cysteine sulfenic acid of the oxidized form is far more stable than that of other peroxiredoxins [17]. The physiological electron donor for 1-Cys peroxiredoxins remains largely unknown.
We isolated previously a 1-Cys peroxiredoxin from bovine eye tissue, which contains one cysteine residue per molecule [18], [19]. In subsequent studies, we found that a recombinant protein of the bovine eye 1-Cys peroxiredoxin (BRPrx) possesses peroxidase activity and is able to protect glutamine synthetase from inactivation by the metal ion-catalyzed oxidation (MCO) system supplemented with dithiothreitol (DTT) as an electron donor [20]. In this study we investigated the reaction of BRPrx with various peroxides and peroxynitrite. Herein, we present evidence that the active center cysteine of BRPrx molecule could exist in two stable, functionally important forms: reduced (Cys-SH) form and oxidized, i.e., sulfenic acid (Cys-SOH) form. We also report on some of the properties of the reduced and oxidized forms of BRPrx active center cysteine.
Section snippets
Materials
L-glutamine synthetase (GS) from Escherichia coli (EC 6.3.1.2) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The supplier’s buffer was replaced by 50 mM HEPES buffer (pH 7.5) by repeated filtration with an Ultrafree-4 centrifugal filter unit (Millipore Corp., Milford, MA, USA). Bovine erythrocyte glutathione peroxidase (GPx) (EC 1.11.1.9) was from Sigma. The supplier’s buffer was replaced by 50 mM phosphate buffer (pH 7.5) containing 0.5 mM EDTA. Horseradish peroxidase (HRPx) (EC
Cysteine is the only amino acid residue oxidized by H2O2
Purification of BRPrx from E. coli extract was carried out in the presence of DTT, which was removed during the last purification step of gel filtration chromatography. The purified BRPrx was tested for the sulfhydryl group using Ellman’s reagent (DTNB) in the absence or presence of denaturing agents. In the native BRPrx no DTNB-reactive cysteine was detected. However, about 0.94 ± 0.03 moles TNB (reaction product between SH-group and DTNB) was generated per mol of BRPrx in the presence of SDS
Discussion
In this study, we found that reduced BRPrx is fairly resistant to oxidation by molecular oxygen but is oxidized rapidly by peroxides and peroxynitrite. The equimolar reaction of BRPrx with these oxidants and loss of its reactivity after modification of the SH-group indicate that the cysteine residue present in the molecule of BRPrx is important for both peroxidase and peroxynitrite reductase activities of the protein and no other amino acid residues are oxidized by peroxides or peroxynitrite.
Acknowledgements
This work was supported by National Institutes of Health Grant EY04694 and a departmental grant from Research to Prevent Blindness, Inc.
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