Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2 •–) and nitric oxide (•NO). In this review, we first ...analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.
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Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it ...promotes nitroxidative damage.
We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.
The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only <1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5μMs−1 and its steady-state concentration at ~1nM.
The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
•Peroxynitrite is a reactive biological oxidant.•Kinetic and mechanistic considerations predict the preferential reactions of peroxynitrite.•Carbon dioxide participates in peroxynitrite decay and oxidations.•Peroxiredoxins represent efficient thiol-based peroxynitrite detoxification systems.•Conceivable biological steady-state concentrations of peroxynitrite can be estimated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors ...such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser–His–Asp triad, proposed to serve a general acid–base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 μmol·kg−1 in brain to 1.4 μmol·kg−1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 μm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Because of the increased insight into the biological role of hydrogen peroxide (H2O2) under physiological and pathological conditions and the role it presumably plays in the action of natural and ...synthetic redox-active drugs, there is a need to accurately define the type and magnitude of reactions that may occur with this intriguing and key species of redoxome. Historically, and frequently incorrectly, the impact of catalase-like activity has been assigned to play a major role in the action of many redox-active drugs, mostly SOD mimics and peroxynitrite scavengers, and in particular MnTBAP3– and Mn salen derivatives. The advantage of one redox-active compound over another has often been assigned to the differences in catalase-like activity. Our studies provide substantial evidence that Mn(III) N-alkylpyridylporphyrins couple with H2O2 in actions other than catalase-related. Herein we have assessed the catalase-like activities of different classes of compounds: Mn porphyrins (MnPs), Fe porphyrins (FePs), Mn(III) salen (EUK-8), and Mn(II) cyclic polyamines (SOD-active M40403 and SOD-inactive M40404). Nitroxide (tempol), nitrone (NXY-059), ebselen, and MnCl2, which have not been reported as catalase mimics, were used as negative controls, while catalase enzyme was a positive control. The dismutation of H2O2 to O2 and H2O was followed via measuring oxygen evolved with a Clark oxygen electrode at 25°C. The catalase enzyme was found to have kcat(H2O2)=1.5×106M–1 s–1. The yield of dismutation, i.e., the maximal amount of O2 evolved, was assessed also. The magnitude of the yield reflects an interplay between the kcat(H2O2) and the stability of compounds toward H2O2-driven oxidative degradation, and is thus an accurate measure of the efficacy of a catalyst. The kcat(H2O2) values for 12 cationic Mn(III) N-substituted (alkyl and alkoxyalkyl) pyridylporphyrin-based SOD mimics and Mn(III) N,N′-dialkylimidazolium porphyrin, MnTDE-2-ImP5+, ranged from 23 to 88M–1 s–1. The analogous Fe(III) N-alkylpyridylporphyrins showed ~10-fold higher activity than the corresponding MnPs, but the values of kcat(H2O2) are still ~4 orders of magnitude lower than that of the enzyme. While the kcat(H2O2) values for Fe ethyl and n-octyl analogs were 803.5 and 368.4M–1 s–1, respectively, the FePs are more prone to H2O2-driven oxidative degradation, therefore allowing for similar yields in H2O2 dismutation as analogous MnPs. The kcat(H2O2) values are dependent on the electron deficiency of the metal site as it controls the peroxide binding in the first step of the dismutation process. SOD-like activities depend on electron deficiency of the metal site also, as it controls the first step of O2●− dismutation. In turn, the kcat(O2●−) parallels the kcat(H2O2). Therefore, the electron-rich anionic non-SOD mimic MnTBAP3– has essentially very low catalase-like activity, kcat(H2O2)=5.8M–1 s–1. The catalase-like activities of Mn(III) and Fe(III) porphyrins are at most, 0.0004 and 0.05% of the enzyme activity, respectively. The kcat(H2O2) values of 8.2 and 6.5M–1 s–1 were determined for electron-rich Mn(II) cyclic polyamine-based compounds, M40403 and M40404, respectively. The EUK-8, with modest SOD-like activity, has only slightly higher kcat(H2O2)=13.5M–1 s–1. The biological relevance of kcat(H2O2) of MnTE-2-PyP5+, MnTDE-2-ImP5+, MnTBAP3–, FeTE-2-PyP5+, M40403, M40404, and Mn salen was evaluated in wild-type and peroxidase/catalase-deficient E. coli.
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•Catalase-like activity of different classes of redox active compounds, described by kcat(H2O2) and yield of O2 evolution, was assessed.•Mn(III) and Fe(III) porphyrins have catalase-like activity in the range of 0.0004 to 0.05% of the enzyme activity, respectively.•Mn salen EUK-8 has 0.0009% of enzyme activity, while M40403, M40404, and tempol have no detectable catalase-like activity.•Low catalase-like activity of compounds studied must be taken into account when drawing conclusions on their mechanism(s) of action(s) in vitro and in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H2S. The presence of a heme cofactor in CBS is enigmatic, ...and its conversion from the ferric– to ferrous–CO state inhibits enzyme activity. The low heme redox potential (−350 mV) has raised questions about the feasibility of the ferrous–CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H2S, via heme-mediated allosteric regulation of CBS.
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Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2 •–). O2 •– reacts at diffusional rates with nitric oxide to yield a potent ...nitrating species, peroxynitrite anion (ONOO–). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0 × 105 M–1 s–1 for the reaction of hMnSOD with ONOO– by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO–, including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO– at a rate of ∼1 × 106 M–1 s–1 to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of ∼1.0 × 105 M–1 s–1, fully consistent with the direct method. Proteomic analysis indicated that ONOO–, but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO– with MnIII to yield MnIV and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO– and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.
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Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal ...phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2 •–) that exists in rapid equilibrium with S2O4 2–. Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10–3 s–1 (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 102 M–1 s–1. Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed a hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H2S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H2S, and superoxide signaling pathways.
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Protein sulfenic acids (R-SOH) are receiving increased interest as intermediates in redox processes. Human serum albumin, the most abundant protein in plasma, possesses a single free thiol. We ...describe herein the different methodologies that we have employed to study the formation of sulfenic acid in this protein and characterize some of its properties, including reactions that lead to the formation of mixed disulfides and the sulfinic acid derivative. The thiol of albumin is oxidized by hydrogen peroxide and peroxynitrite to a relatively stable sulfenic acid, which can be detected through different strategies including reduction with sodium arsenite and reaction with glutathione. Dimedone trapping followed by mass spectrometry analysis confirmed the modification. The challenge of obtaining quantitative data regarding albumin sulfenic acid has been approached using the yellow thiol thionitrobenzoate. A careful analysis has led to the determination of the rate constants of the reactions of sulfenic acid with analytical probes and with possible biological targets such as plasma thiols, which lead to mixed disulfides, and hydrogen peroxide, which overoxidizes the sulfenic to sulfinic acid. Our results support the concept that sulfenic acid is a central intermediate in the formation of oxidized albumin species that are present in circulating albumin and increase under pathological conditions.
Cationic manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can ...reach mitochondria offering protection against reactive species in different animal models of disease. Although an LC-MS/MS-based method for MnP quantitation and subcellular distribution has been reported, a direct method capable of evaluating both the uptake and the redox state of MnP in living cells has not yet been developed. In the present work we applied resonance Raman (RR) spectroscopy to analyze the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ within endothelial cells. RR experiments with isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. To assess the combined impact of reactivity and bioavailability, we studied the kinetics of Mn(III)TnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.
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•Raman Resonance was utilized to reveal the redox state of Mn-porphyrins (MnP) in cells.•Mn(III)P are readily reduced intracellularly to the Mn(II) state.•Intramitochondrial oxidation of a peroxynitrite-sensitive probe is inhibited by MnP.•The cytotoxicity of peroxynitrite is neutralized by MnP via a catalytic redox cycle.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are ...scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA−SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was ∼20-fold faster than the second phase and first order in HSA−SOH and TNB (105 ± 11 M-1 s-1, 25 °C, pH 7.4), allowing quantitative data on HSA−SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 °C, 4 min) yielded 0.18 ± 0.02 mol of HSA−SOH per mol of HSA. HSA−SH reacted with hydrogen peroxide at 2.7 ± 0.7 M-1 s-1 (37 °C, pH 7.4), while HSA−SOH reacted at 0.4 ± 0.2 M-1 s-1, yielding sulfinic acid (HSA−SO2H), as detected by mass spectrometry. The rate constants of HSA−SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA−SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA−SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 ± 0.2, 2.9 ± 0.5, 9.3 ± 0.9, and 55 ± 3 M-1 s-1 (25 °C, pH 7.4), respectively, supporting a role for HSA−SOH in the formation of mixed disulfides.
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