Elsevier

Free Radical Biology and Medicine

Volume 89, December 2015, Pages 1049-1056
Free Radical Biology and Medicine

Original Contribution
Kinetics of reaction of peroxynitrite with selenium- and sulfur-containing compounds: Absolute rate constants and assessment of biological significance

https://doi.org/10.1016/j.freeradbiomed.2015.10.424Get rights and content

Highlights

  • Peroxynitrous acid (ONOOH) is a major damaging oxidant formed during inflammation

  • ONOOH targets sulfur species, and was hypothesized to also target selenium centers.

  • Rate constants for ONOOH with selenols (RSeH) are 250–830 fold higher than thiols.

  • Selenomethionine is ~13-fold more reactive than methionine.

  • Selenium compounds are major targets for ONOOH at extracellular sites.

Abstract

Peroxynitrite (the physiological mixture of ONOOH and its anion, ONOO) is a powerful biologically-relevant oxidant capable of oxidizing and damaging a range of important targets including sulfides, thiols, lipids, proteins, carbohydrates and nucleic acids. Excessive production of peroxynitrite is associated with several human pathologies including cardiovascular disease, ischemic-reperfusion injury, circulatory shock, inflammation and neurodegeneration. This study demonstrates that low-molecular-mass selenols (RSeH), selenides (RSeR') and to a lesser extent diselenides (RSeSeR') react with peroxynitrite with high rate constants. Low molecular mass selenols react particularly rapidly with peroxynitrite, with second order rate constants k2 in the range 5.1×105–1.9×106 M−1 s−1, and 250–830 fold faster than the corresponding thiols (RSH) and many other endogenous biological targets. Reactions of peroxynitrite with selenides, including selenosugars are approximately 15-fold faster than their sulfur homologs with k2 approximately 2.5×103 M−1 s−1. The rate constants for diselenides and sulfides were slower with k2 0.72–1.3×103 M−1 s−1 and approximately 2.1×102 M−1 s−1 respectively. These studies demonstrate that both endogenous and exogenous selenium-containing compounds may modulate peroxynitrite-mediated damage at sites of acute and chronic inflammation, with this being of particular relevance at extracellular sites where the thiol pool is limited.

Introduction

Peroxynitrous acid (ONOOH) is a potent oxidant and nitrating species that is formed in vivo and in vitro by the cross-reaction (termination) of two biologically-important radicals: superoxide (O2•−; generated by a respiratory burst from activated leukocytes) and nitric oxide (NO; generated by nitric oxide synthase enzymes present in multiple cells) [1], [2], [3]. This species, and to a lesser extent its anion ONOO (pKa ~6.8, though this is buffer dependent [4], [5]), oxidizes a wide variety of biomolecules including thioethers (such as methionine [6]), thiols [7], lipids [8], [9], proteins (e.g. [10], [11]), carbohydrates [12] and nucleic acids [13], [14], [15] either through direct two-electron oxidation, or via limited generation of reactive secondary radicals such as HO and NO2 [16], [17], [18]. In the presence of bicarbonate (HCO3) additional reactions involving the peroxynitrosocarbonate anion (ONOOCO2), and radicals derived from homolysis of this species (HO and CO3) also play a role in inducing oxidative damage [17].

Excessive, mistimed or misplaced production of peroxynitrite (the physiological mixture of ONOOH and ONOO) has been associated with the development of organ damage and dysfunction in a number of pathologies including cardiovascular disease [19], [20], [21], ischemia-reperfusion injury [22], [23], liver damage [24], circulatory shock [23], central nervous system damage [25], and neurodegeneration [26], [27], amongst others. In such circumstances endogenous cellular and extracellular defense mechanisms may be overwhelmed, and synthetic materials that react rapidly with peroxynitrite may represent novel pharmacological protective agents for such conditions [28]. A number of low-molecular-mass compounds have been postulated as biological targets for, or protective agents against, peroxynitrite-mediated damage including thiols (e.g. glutathione, GSH), ascorbate, carbon dioxide (CO2) and synthetic metalloporphyrins [29], [30], [31], [32]. Additional studies have shown that reaction of peroxynitrite with some proteins including peroxidases, peroxiredoxins, hemoglobin, albumin and some selenoproteins is very rapid [29], [30], [31], [32], [33], [34]. In the light of the concentrations of these species present in vivo it has been postulated that reaction with CO2 (to give the nitrosoperoxycarbonate anion [35], [36]) and GSH is likely to be quantitatively the most significant [32], though kinetic data for the reactions of peroxynitrite particularly with seleno-species are incomplete.

Selenium, in the form of selenocysteine (Sec), a RNA coded amino acid, plays a critical role in the activity of a number of cellular protective enzymes including glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and some isoforms of methionine sulfoxide reductase [37], [38]. GPx in conjunction with its co-factor, glutathione (GSH), catalyzes the reduction of H2O2 (and for GPx4, lipid hydroperoxides [39]) and peroxynitrite [40]. The reactions of GPx and peroxiredoxins (and related species) with peroxynitrite have been postulated to occur via protein-bound Sec and Cys residues respectively, with second order rate constants (k2) in the range 105–107 M−1 s−1 (e.g. [30], [33], [34], [41], [42], [43], [44]). However it is unclear whether the high reactivity of these residues, and particularly Sec, is an inherent property of the free amino acid (and related seleno compounds), or a function of the surrounding protein. The synthetic selenoamide ebselen, and the natural amino acid selenomethionine (SeMet) are known to react more rapidly with peroxynitrite than their sulfur analogs, though k2 for these low-molecular-mass species are, at least for SeMet, considerably slower (~1.5×103 M−1 s−1 [45]) than for the Sec residue of GPx. We have recently reported novel selenium-containing carbohydrates that react with HOX (X=Cl, Br, SCN) with high rate constants, and protect both isolated and plasma proteins from hypochlorous acid (HOCl)- and chloramine-mediated damage [46], [47], [48] (reviewed [49]). In the light of the high reactivity of these seleno compounds with HOCl we hypothesized that low-molecular-mass selenols, seleno sugars, and diselenides would also react rapidly with peroxynitrite. The absolute rate constants reported here for these reactions support this hypothesis. These rate constants and those for their sulfur analogs obtained for reaction with peroxynitrite, are also compared to other oxidants to allow the biological significance of these reactions and the role of structure in determining the reaction kinetics to be assessed.

Section snippets

Materials

3,3′-Diselenodipropionic acid was synthesized and purified as described previously [50]. The selenosugars were synthesized as described previously [46], [47]. The diselenide of selenocysteine methyl ester [-SeCH2CH(NH2)C(=O)OCH3]2 was synthesized as reported previously [51]. All other chemicals were from Sigma-Aldrich/Fluka and used without further purification. All selenium-containing compounds were dissolved in Chelex-treated sodium phosphate buffer (pH 7.4, 200 mM) prepared using Milli Q

Determination of rate constants for reaction of peroxynitrite with low-molecular-mass selenols

Selenols (RSeH) are structurally similar to thiols (RSH), but have lower pKa values (i.e. are stronger acids) and exist primarily in their anion form (RSe, pKa 5.2 for Sec) at neutral pH [58]. Selenols are powerful nucleophiles and undergo ready oxidation in air [58], so these materials were synthesized in their diselenide (RSeSeR') forms. The selenols were obtained from the diselenides for kinetic studies by in situ reduction with sodium borohydride [55]. The slow decay of the stock selenol

Discussion

Peroxynitrite is a major oxidant generated at sites of inflammation that is both bactericidal and potentially damaging to host tissues (reviewed: [16], [17], [59]). Peroxynitrite can react directly and rapidly, with proteins, metal ions, selenium-containing proteins and pharmaceuticals, and (via its anion form ONOO) with CO2 to give the peroxynitrosocarbonate anion (ONOOCO2) [11], [17], [59], [60]. Homolytic cleavage of both ONOOH and ONOOCO2 can result in radical formation and additional

Acknowledgments

We are grateful to the Australian Research Council (through the Centres of Excellence Scheme, CE0561607, and Discovery Programs DP0988311), the National Heart Foundation of Australia (Grants-in-aids G09S4313 and G11S 5811) and the Novo Nordisk Foundation (Laureate Research Grant NNF13OC0004294 to MJD) for financial support. We thank Professors Vimal K. Jain and K. Indira Priyadarsini of Bhabha Atomic Research Centre, Mumbai (India), for providing 3,3′-diselenodipropionic acid.

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