Péter Nagy

Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: implications of their possible biological activity and utility.

Hydrogen sulfide (H2S) is an endogenously generated and putative signaling/effector molecule. Despite its numerous reported functions, the chemistry by which it elicits its functions is not understood. Moreover, recent studies allude to the existence of other sulfur species besides H2S that may play critical physiological roles. Herein, the basic chemical biology of H2S as well as other related or derived species is discussed and reviewed. This review particularly focuses on the per- and polysulfides which are likely in equilibrium with free H2S and which may be important biological effectors themselves.

Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study.

Peroxiredoxins (Prx) are thiol peroxidases that exhibit exceptionally high reactivity toward peroxides, but the chemical basis for this is not well understood. We present strong experimental evidence that two highly conserved arginine residues play a vital role in this activity of human Prx2 and Prx3. Point mutation of either ArgI or ArgII (in Prx3 Arg-123 and Arg-146, which are ∼3–4 Å or ∼6–7 Å away from the active site peroxidative cysteine (Cp), respectively) in each case resulted in a 5 orders of magnitude loss in reactivity. A further 2 orders of magnitude decrease in the second-order rate constant was observed for the double arginine mutants of both isoforms, suggesting a cooperative function for these residues. Detailed ab initio theoretical calculations carried out with the high level G4 procedure suggest strong catalytic effects of H-bond-donating functional groups to the Cp sulfur and the reactive and leaving oxygens of the peroxide in a cooperative manner. Using a guanidinium cation in the calculations to mimic the functional group of arginine, we were able to locate two transition structures that indicate rate enhancements consistent with our experimentally observed rate constants. Our results provide strong evidence for a vital role of ArgI in activating the peroxide that also involves H-bonding to ArgII. This mechanism could explain the exceptional reactivity of peroxiredoxins toward H2O2 and may have wider implications for protein thiol reactivity toward peroxides.

Kinetics and Mechanisms of the Reaction of Hypothiocyanous Acid with 5-Thio-2-nitrobenzoic Acid and Reduced Glutathione

Hypothiocyanite is a major oxidant generated by mammalian peroxidases. Although reported to react specifically with thiol groups in biological molecules, a detailed mechanistic study of this reaction has not been conducted. We have investigated the reaction of hypothiocyanous acid/hypothiocyanite with 5-thio-2-nitrobenzoic acid and with reduced glutathione by stopped-flow spectroscopy. The observed bell-shaped pH profile established that the reaction with 5-thio-2-nitrobenzoic acid proceeds via the thiolate and hypothiocyanous acid in the 2.5 < pH < 8 region. The obtained second-order rate constant of the reaction is (1.26 + or - 0.02) x 10(8) M(-1) s(-1), and the effective rate constant at pH 7.4 is (4.37 + or - 0.03) x 10(5) M(-1) s(-1). Analysis of the kinetic data, using a value of 4.38 + or - 0.01 for the pK(a) of 5-thio-2-nitrobenzoic acid thiol (measured independently by spectroscopic analysis), gave a pK(a) of 4.85 + or - 0.01 for hypothiocyanous acid at physiological salt concentration (I = 120 mM; NaCl and phosphate buffer) and 25 degrees C. A second-order rate constant of (8.0 + or - 0.5) x 10(4) M(-1) s(-1) for the reaction of hypothiocyanous acid/hypothiocyanite with reduced glutathione at pH 7.4 was determined. The glutathione data are also consistent with the reaction proceeding via the thiolate and hypothiocyanous acid. Our results demonstrate that hypothiocyanous acid/hypothiocyanite has very high reactivity with thiols and will be short-lived in the presence of physiological concentrations of glutathione and thiol proteins. As the reaction occurs strictly with the thiolate, this oxidant should selectively target proteins containing low pK(a) thiols.

Redox Chemistry of Biological Thiols

Publisher Summary This chapter discusses two-electron oxidations of thiols and radical-mediated reactions. It demonstrates the importance of the oxidizing and electrophilic properties of the oxidant as well as the nucleophilicity of the sulfur center in these reactions. The chemical properties of sulfur make it very reactive in biological systems. It has strong electrondonating and electron-accepting properties and an extraordinary reactivity compared to oxygen or nitrogen. The thiol functional group (–SH also called a sulfhydryl group), where the sulfur atomis in its lowest oxidation state, is the functional group of the amino acid cysteine. Thiols engage in two-electron oxidation reactions and are also efficient scavengers of radical species. The rates of these reactions are dependent on the chemical properties of the oxidant as well as the thiol. The ionizability of the sulfhydryl group is a major determining factor in its nucleophilic character. The nucleophilic character of thiols is probably the most important factor that determines reactivity. This property is reflected in thiol ionizability and, therefore, the acid dissociation constants of sulfhydryl groups provide invaluable information in kinetic studies. In addition the chapter also highlights the physiological importance and the redox properties of hydrogen sulfide (H 2 S), a newly recognized signaling molecule that can be seen from the chemical point of view as the smallest thiol.

Superoxide-mediated Formation of Tyrosine Hydroperoxides and Methionine Sulfoxide in Peptides through Radical Addition and Intramolecular Oxygen Transfer

The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and have major implications.

Removal of amino acid, peptide and protein hydroperoxides by reaction with peroxiredoxins 2 and 3

Prxs (peroxiredoxins) are a ubiquitous family of cysteine-dependent peroxidases that react rapidly with H2O2 and alkyl hydroperoxides and provide defence against these reactive oxidants. Hydroperoxides are also formed on amino acids and proteins during oxidative stress, and they too are a potential cause of biological damage. We have investigated whether Prxs react with amino acid, peptide and protein hydroperoxides, and whether the reactions are sufficiently rapid for these enzymes to provide antioxidant protection against these oxidants. Isolated Prx2, which is a cytosolic protein, and Prx3, which resides within mitochondria, were reacted with a selection of hydroperoxides generated by γ-radiolysis or singlet oxygen, on free amino acids, peptides and proteins. Reactions were followed by measuring the accumulation of disulfide-linked Prx dimers, via non-reducing SDS/PAGE, or the loss of the corresponding hydroperoxide, using quench-flow and LC (liquid chromatography)/MS. All the hydroperoxides induced rapid oxidation, with little difference in reactivity between Prx2 and Prx3. N-acetyl leucine hydroperoxides reacted with Prx2 with a rate constant of 4 × 10(4) M-1 · s-1. Hydroperoxides present on leucine, isoleucine or tyrosine reacted at a comparable rate, whereas histidine hydroperoxides were ~10-fold less reactive. Hydroperoxides present on lysozyme and BSA reacted with rate constants of ~100 M-1 · s-1. Addition of an uncharged derivative of leucine hydroperoxide to intact erythrocytes caused Prx2 oxidation with no concomitant loss in GSH, as did BSA hydroperoxide when added to concentrated erythrocyte lysate. Prxs are therefore favoured intracellular targets for peptide/protein hydroperoxides and have the potential to detoxify these species in vivo.

Reactions of superoxide with the myoglobin tyrosyl radical.

The contribution of superoxide-mediated injury to oxidative stress is not fully understood. A potential mechanism is the reaction of superoxide with tyrosyl radicals, which either results in repair of the tyrosine or formation of tyrosine hydroperoxide by addition. Whether these reactions occur with protein tyrosyl radicals is of interest because they could alter protein structure or modulate enzyme activity. Here, we have used a xanthine oxidase/acetaldehyde system to generate tyrosyl radicals on sperm whale myoglobin in the presence of superoxide. Using mass spectrometry we found that superoxide prevented myoglobin dimer formation by repairing the protein tyrosyl radical. An addition product of superoxide at Tyr151 was also identified, and exogenous lysine promoted the formation of this product. In our system, reaction of tyrosyl radicals with superoxide was favored over dimer formation with the ratio of repair to addition being approximately 10:1. Our results demonstrate that reaction of superoxide with protein tyrosyl radicals occurs and may play a role in free radical-mediated protein injury.

Hypothiocyanous acid is a potent inhibitor of apoptosis and caspase 3 activation in endothelial cells.

Hypothiocyanous acid (HOSCN) is a common, thiol-specific oxidant with strong antibacterial activity. It is thought to be nontoxic to mammalian cells, although its ability to specifically target intracellular thiols may potentially cause cellular dysfunction. In this study we demonstrate specific effects of HOSCN on human endothelial cells, with exposure to high concentrations resulting in morphology changes unlike those seen with other oxidants. Effects were time- and dose-dependent and were accompanied by loss of total cell thiols and GSH and by inactivation of glyceraldehyde-3-phosphate dehydrogenase. High-dose exposure was cytotoxic, but lesser doses did not cause cell death, and apoptosis was not initiated by any concentration of HOSCN. In fact, initiation of apoptosis was blocked by minimal HOSCN exposure, with activation of caspase 3 and cleavage of the proenzyme being prevented. This was unlikely to be due to direct oxidation of the caspase 3 active-site cysteine and suggests alternative targeting of the caspase pathway. The survival of endothelial cells when HOSCN is present together with an inducer of apoptosis suggests that HOSCN differs from most other oxidants and could affect endothelial cell survival pathways in a way that may have an impact on vascular function.

Rapid reaction of superoxide with insulin-tyrosyl radicals to generate a hydroperoxide with subsequent glutathione addition.

Tyrosine (Tyr) residues are major sites of radical generation during protein oxidation. We used insulin as a model to study the kinetics, mechanisms, and products of the reactions of radiation-induced or enzyme-generated protein-tyrosyl radicals with superoxide to demonstrate the feasibility of these reactions under oxidative stress conditions. We found that insulin-tyrosyl radicals combined to form dimers, mostly via the tyrosine at position 14 on the α chain (Tyr14). However, in the presence of superoxide, dimerization was largely outcompeted by the reaction of superoxide with insulin-tyrosyl radicals. Using pulse radiolysis, we measured a second-order rate constant for the latter reaction of (6±1) × 10(8) M(-1) s(-1) at pH 7.3, representing the first measured rate constant for a protein-tyrosyl radical with superoxide. Mass-spectrometry-based product analyses revealed the addition of superoxide to the insulin-Tyr14 radical to form the hydroperoxide. Glutathione efficiently reduced the hydroperoxide to the corresponding monoxide and also subsequently underwent Michael addition to the monoxide to give a diglutathionylated protein adduct. Although much slower, conjugation of the backbone amide group can form a bicyclic Tyr-monoxide derivative, allowing the addition of only one glutathione molecule. These findings suggest that Tyr-hydroperoxides should readily form on proteins under oxidative stress conditions where protein radicals and superoxide are both generated and that these should form addition products with thiol compounds such as glutathione.

Neutrophil-mediated oxidation of enkephalins via myeloperoxidase-dependent addition of superoxide.

Neutrophils play a major role in acute inflammation in part by generating superoxide and an array of other reactive species. These white blood cells also contribute to protection against inflammatory pain by releasing opioid peptides. The biochemical interactions of enkephalins with neutrophil-derived oxidants are not well understood. In this investigation we reveal that neutrophils use myeloperoxidase to oxidize enkephalins to their corresponding tyrosyl free radicals, which react preferentially with the superoxide to form a hydroperoxide. In methionine enkephalin, rapid intramolecular oxygen transfer from the hydroperoxide to the Met sulfur results in the formation of a sulfoxide derivative. This reaction may occur at sites of inflammation where enkephalins are released and neutrophils generate large amounts of superoxide. Hydroperoxide formation destroys the aromatic character of the Tyr residue by forming a bicyclic structure via conjugate addition of the terminal amine to the phenol ring. As the N-terminal Tyr and its amino group are essential for their opiate activity, we hypothesize that oxidative modification of this residue should affect the analgesic activity of enkephalins.