Victor S. Sharov

Glutathione Peroxidase Protects against Peroxynitrite-mediated Oxidations A NEW FUNCTION FOR SELENOPROTEINS AS PEROXYNITRITE REDUCTASE

There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing enzymes catalyzing peroxynitrite reduction using glutathione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by peroxynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2 × 106 m −1 s−1. Carboxymethylation of selenocysteine in GPx by iodoacetate led to the loss of “classical” glutathione peroxidase activity but maintained protection against peroxynitrite-mediated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 μmsteady-state concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause the loss of classical GPx activity. GPx, like selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethionine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite-mediated oxidations, as a peroxynitrite reductase.

Protection against peroxynitrite by selenoproteins.

Abstract Cellular defense against excessive peroxynitrite generation is required to protect against DNA strand-breaks and mutations and against interference with protein tyrosine-based sig naling and other protein functions due to formation of 3-nitrotyrosine. We recently demon strated a role of selenium-containing enzymes catalyzing peroxynitrite reduction. Glutathione peroxidase (GPx) protected against the oxidation of dihydrorhodamine 123 (D H R) by perox ynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoor-ganic compound exhibiting a high second-order rate constant for the reaction with peroxynit rite, 2 × 106 M-1 S-1. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. Similarly, selenomethionine but not selenomethionine oxide exhibited inhibition of rhodamine 123 formation from DHR caused by peroxynitrite. In steady-state experiments, in which peroxynitrite was infused to maintain a 0.2 μᴍ con centration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhib ited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause loss of ‘classical’ GPx activity. GPx, like selenomethionine, pro tected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx , ebselen or selenomethionine. The selenoxides can be effectively reduced by glutathione, establishing a biological line of defense against peroxynitrite. The novel function of GPx as a peroxynitrite reductase may extend to other selenoproteins containing selenocysteine or selenomethionine. Recent work on organotellurium compounds revealed peroxynitrite reductase activity as well. Inhibition of dihydrorhodamine 123 oxidation correlated well with the GPx-like activity of a variety of diaryl tellurides.

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation.

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB‐F344 rat liver epithelial cells, assayed by the scrapeloading dye‐transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady‐state concentration of peroxynitrite of 1.6 muM for 4 min or to 3‐morpholinosydnonimine (SIN‐1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN‐1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1‐1 muM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium‐containing proteins protect.

Sensitization of Peroxynitrite Chemiluminescence by the Triplet Carbonyl Sensitizer Coumarin‐525. Effect of CO2

Abstract— The flash of spontaneous chemiluminescence (CL) that reflects the formation of electronically excited intermediates during the decay of peroxynitrite (ONOO) to nitrate was investigated. The half‐decay time of the CL flash (0.5 ± 0.1 s) was in agreement with the half‐life of peroxynitrite obtained in stopped‐flow experiments. The spontaneous CL intensity was linearly dependent on peroxynitrite concentration. The yield of spontaneous CL from peroxynitrite decay, 2 × 10‐9 photons/peroxynitrite at pH 9.5, was strongly enhanced by a sensitizer of triplet carbonyl CL, coumarin‐525 (C‐525). The maximal yield of sensitized CL was calculated to be 3 × 10‐6 photons/ peroxynitrite molecule for infinite concentration of C‐525. The dependence of both spontaneous and sensitized CL on pH has a maximum at about pH 9.5. Bubbling with CO2 or addition of NaHCO3 considerably enhanced the flash of CL, and it is concluded that the reaction of the peroxynitrite anion with CO2 is a major pathway leading to the formation of an electronically excited intermediate of peroxynitrite.

Electronically excited intermediate from peroxynitrite: evaluation by chemiluminescence and by the isomerization of β-carotene

A set of hydrophobic triplet energy acceptors, chemiluminescence (CL) sensitizers and β-carotene, has been used to evaluate electronically excited intermediate formation during peroxynitrite (ONOO−) decay to nitrate in tetrahydrofuran. The yield of spontaneous CL (ΦCL = 1.3 × 10−9 photons/peroxynitrite) in tetrahydrofuran is close to the value obtained for aqueous solution. CL is strongly enhanced by sensitizers of triplet carbonyl chemiluminescence, coumarin 525 and chlorophyll-a, most likely by the energy transfer mechanism. Maximal yields of sensitized CL have been calculated to be 10−6 and 2 × 10−6 photons/peroxynitrite, respectively. The acceptor of high-energy triplets, 9,10-dibromoanthracene, is a weak sensitizer, so the energy of the electronically excited intermediate of peroxynitrite is within the range 56 ≤ ΔE ≤ 67 kcal mol−1. The yield of the excited intermediate of peroxynitrite in tetrahydrofuran is estimated from sensitized chemiluminescence to be about 0.2 mole per mole of peroxynitrite decomposed. This estimation is in accordance with the observed maximal yields of all-trans- and 13-cis-β-carotene trans/cis or cis/trans isomerization, which is believed to proceed via energy transfer from the excited intermediate of peroxynitrite to β-carotene, 0.06 and 0.14 (mole of isomerized β-carotene per mole of peroxynitrite), respectively. These results indicate that an electronically excited intermediate of peroxynitrite is produced with considerable yield and may be involved in peroxynitrite-mediated reactions, e.g., β-carotene isomerization.

Sensitized Chemiluminescence and Fluorescence Methods in Studies of Oxidative Stress

Oxidative stress is one of the pathogenic mechanisms involved in cell injury. Various reactive species can be formed during oxidative reactions. Electronically excited molecules such as excited carbonyls or singlet oxygen can directly generate chemiluminescence in biological systems (Cilento 1982; Cadenas and Sies 1984). Excited carbonyls are formed during peroxidation of unsaturated fatty acids by disproportionation of two peroxyl radicals through the Russell mechanism or by disproportionation of alkoxyl radicals or by decomposition of dioxetanes (Russell 1957; Cadenas 1989). In biological systems triplet carbonyls cause weak emission and can provide indirect evidence for the generation of peroxyl radicals (Noll et al. 1987). Singlet oxygen can be monitored in the red region of visible light at 634 nm and 703 nm (dimol emission) or in the infrared region at 1270 nm (monomol emission).

6 – A New Function for Selenoproteins: Peroxynitrite Reduction

Publisher Summary Selenoproteins carry out a variety of catalytic functions, many of which are redox reactions. A novel function for selenoproteins that has been reported is the reduction of peroxynitrite. Studies were prompted by the observation of a very efficient reduction of peroxynitrite by ebselen, exhibiting the highest second-order rate constant for a low-molecular-weight compound with peroxynitrite known so far, 2.0 x 106 M-lsec-1. In analogy to the reaction cycle for ebselen, Scheme 1A presents the proposed sequence. In the first step, the selenocysteine, probably as the selenolate, reacts with peroxynitrite to oxidize to the corresponding selenenic acid, yielding nitrite. However, peroxynitrous acid may also react to yield nitrous acid. The subsequent two steps in the reaction cycle are facile regeneration reactions at the expense of reducing equivalents provided by GSH in cells, known from the extensive work on GPx. Regarding the chemical mechanism, it might be concluded that the selenolate form of selenocysteine residue is required. However, a selenol moiety is not strictly necessary for peroxynitrite reductase activity, in contrast to the GSH peroxidase action, because the carboxymethylated selenium derivative maintained activity. This is in accord with the high rate constant obtained for 2-(methylseleno) benzanilide and for selenomethionine (Scheme 1B).