Superoxide Flux Research Articles

Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO−) and superoxide (HO2•/O2•−). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO−. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.

Nanoceria potently reduce superoxide fluxes from mitochondrial electron transport chain and plasma membrane NADPH oxidase in human macrophages.

Cerium oxide nanoparticles, also known as nanoceria, possess antioxidative and anti-inflammatory activities in animal models of inflammatory disorders, such as sepsis. However, it remains unclear how nanoceria affect cellular superoxide fluxes in macrophages, a critical type of cells involved in inflammatory disorders. Using human ML-1 cell-derived macrophages, we showed that nanoceria at 1-100μg/ml potently reduced superoxide flux from the mitochondrial electron transport chain (METC) in a concentration-dependent manner. The inhibitory effects of nanoceria were also shown in succinate-driven mitochondria isolated from the macrophages. Furthermore, nanoceria markedly mitigated the total intracellular superoxide flux in the macrophages. These data suggest that nanoceria could readily cross the plasma membrane and enter the mitochondrial compartment, reducing intracellular superoxide fluxes in unstimulated macrophages. In macrophages undergoing respiratory burst, nanoceria also strongly reduced superoxide flux from the activated macrophage plasma membrane NADPH oxidase (NOX) in a concentration-dependent manner. Token together, the results of the present study demonstrate that nanoceria can effectively diminish superoxide fluxes from both METC and NOX in human macrophages, which may have important implications for nanoceria-mediated protection against inflammatory disease processes.

Study of the relationship between extracellular superoxide and glutathione production in batch cultures of Escherichia coli

Aerobically growing Escherichia coli generates superoxide flux into the periplasm via the oxidation of dihydromenaquinone and simultaneously carries out continuous transmembrane cycling of glutathione (GSH). Here we have shown that, under the conditions of a gradual decrease in dissolved oxygen (dO2), characteristic of batch culture, the global regulatory system ArcB/ArcA can play an important role in the coordinated control of extracellular superoxide and GSH fluxes and their interaction with intracellular antioxidant systems. The lowest superoxide production was observed in the menA and arcB mutants, while the atpA, atpC and atpE mutants generated superoxide 1.3–1.5 times faster than the parent. The share of exported glutathione in the ubiC, atpA, atpC, and atpE mutants was 2–3 times higher compared to the parent. A high direct correlation (r = 0.87, p = 0.01) between extracellular superoxide and GSH was revealed. The menA and arcB mutants, as well as the cydD mutant lacking the GSH export system CydDC, were not capable of GSH excretion with a decrease in dO2, which indicates a positive control of GSH export by ArcB. In contrast, ArcB downregulates sodA, therefore, an inverse correlation (r = −0.86, p = 0.013) between superoxide production and sodA expression was observed.

Dark biological superoxide production as a significant flux and sink of marine dissolved oxygen.

The balance between sources and sinks of molecular oxygen in the oceans has greatly impacted the composition of Earth's atmosphere since the evolution of oxygenic photosynthesis, thereby exerting key influence on Earth's climate and the redox state of (sub)surface Earth. The canonical source and sink terms of the marine oxygen budget include photosynthesis, respiration, photorespiration, the Mehler reaction, and other smaller terms. However, recent advances in understanding cryptic oxygen cycling, namely the ubiquitous one-electron reduction of O2 to superoxide by microorganisms outside the cell, remains unexplored as a potential player in global oxygen dynamics. Here we show that dark extracellular superoxide production by marine microbes represents a previously unconsidered global oxygen flux and sink comparable in magnitude to other key terms. We estimate that extracellular superoxide production represents a gross oxygen sink comprising about a third of marine gross oxygen production, and a net oxygen sink amounting to 15 to 50% of that. We further demonstrate that this total marine dark extracellular superoxide flux is consistent with concentrations of superoxide in marine environments. These findings underscore prolific marine sources of reactive oxygen species and a complex and dynamic oxygen cycle in which oxygen consumption and corresponding carbon oxidation are not necessarily confined to cell membranes or exclusively related to respiration. This revised model of the marine oxygen cycle will ultimately allow for greater reconciliation among estimates of primary production and respiration and a greater mechanistic understanding of redox cycling in the ocean.

Correction to: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association.

Correction to: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association.

Potentiating antibiotics in drug-resistant clinical isolates via stimuli-activated superoxide generation.

The rise of multidrug-resistant (MDR) bacteria is a growing concern to global health and is exacerbated by the lack of new antibiotics. To treat already pervasive MDR infections, new classes of antibiotics or antibiotic adjuvants are needed. Reactive oxygen species (ROS) have been shown to play a role during antibacterial action; however, it is not yet understood whether ROS contribute directly to or are an outcome of bacterial lethality caused by antibiotics. We show that a light-activated nanoparticle, designed to produce tunable flux of specific ROS, superoxide, potentiates the activity of antibiotics in clinical MDR isolates of Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Despite the high degree of antibiotic resistance in these isolates, we observed a synergistic interaction between both bactericidal and bacteriostatic antibiotics with varied mechanisms of action and our superoxide-producing nanoparticles in more than 75% of combinations. As a result of this potentiation, the effective antibiotic concentration of the clinical isolates was reduced up to 1000-fold below their respective sensitive/resistant breakpoint. Further, superoxide-generating nanoparticles in combination with ciprofloxacin reduced bacterial load in epithelial cells infected with S. enterica serovar Typhimurium and increased Caenorhabditis elegans survival upon infection with S. enterica serovar Enteriditis, compared to antibiotic alone. This demonstration highlights the ability to engineer superoxide generation to potentiate antibiotic activity and combat highly drug-resistant bacterial pathogens.

Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II.

Melanogenesis is a highly conserved process of cytophotoprotection from UV radiation present in many species. Although both mitochondrial function and UV radiation insults are well-documented promoters of increased cellular stress, their individual molecular relationships with skin pigmentation have not been clearly resolved. This study provides evidence for a direct relationship between cellular melanin content, superoxide flux, and mitochondrial function at complex II. Direct and significant correlation between increased pigmentation and complex II turnover was observed in genetically different melanoma cell lines of varied basal pigmentation states (P < 0.01). The same trend was also observed when comparing genetically identical cell cultures with increasing levels of induced pigmentation (P < 0.005). The observation of increased steady-state levels of the catalytic complex II succinate dehydrogenase subunit A alongside hyperpigmentation suggested coregulation of activity and pigment production (P < 0.01). The study also presents novel evidence for a relationship between hyperpigmentation and increased superoxide-generating capacity at complex II. By amperometrically monitoring superoxide flux from differently pigmented FM55 melanocytes and their isolated mitochondria, a dynamic and responsive relationship between pigmentation, complex II function, and intracellular superoxide generation was observed (P < 0.005). The data support hyperpigmentation as a protective antioxidant mechanism in response to complex II-mediated reactive oxygen species generation.

Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid

Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7-23 M(-1) s(-1)) were significantly higher than that measured for H2O2 (1.5 M(-1) s(-1)). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1-1.5 × 10(3) M(-1) s(-1). Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.

Nitroxidative chemistry interferes with fluorescent probe chemistry: Implications for nitric oxide detection using 2,3-diaminonaphthalene

Simultaneous production of nitric oxide (NO) and superoxide generates peroxynitrite and causes nitroxidative stress. The fluorometric method for NO detection is based on the formation of a fluorescent product from the reaction of a nonfluorescent probe molecule with NO-derived nitrosating species. Here, we present an example of how nitroxidative chemistry could interact with fluorescent probe chemistry. 2,3-Naphthotriazole (NAT) is the NO-derived fluorescent product of 2,3-diaminonaphthalene (DAN), a commonly used NO-detecting molecule. We show that NO/superoxide cogeneration, and particularly peroxynitrite, mediates the chemical decomposition of NAT. Moreover, the extent of NAT decomposition depends on the relative fluxes of NO and superoxide; the maximum effect being reached at almost equivalent generation rates for both radicals. The rate constant for the reaction of NAT with peroxynitrite was determined to be 2.2×103M−1s−1. Further, various peroxynitrite scavengers were shown to effectively inhibit NO/superoxide- and peroxynitrite-mediated decomposition of NAT. Taken together, the present study suggests that the interference of a fluorometric NO assay can be originated from the interaction between the final fluorescent product and the formed reactive nitrogen and oxygen species.

A paradigm shift for local blood flow regulation

a remarkable example of physiological regulation is the coordination between metabolic rate and local blood flow in microscopic volumes of an organ. In skeletal muscle an increase in oxygen consumption (Vo2) over a wide range evokes a proportional (∼5 to 6 × Vo2) increase in blood flow ([29][

Degradation of Perfluorooctanoic Acid by Reactive Species Generated through Catalyzed H2O2 Propagation Reactions

Perfluorinated compounds, which are environmentally persistent and bioaccumulative contaminants, cannot currently be treated in the subsurface by in situ technologies. Catalyzed H2O2 propagation (CHP) reactions, which generate hydroxyl radical, hydroperoxide anion, and superoxide anion, were investigated for treating perfluorooctanoic acid (PFOA) as a basis for in situ chemical oxidation remediation of groundwater. Using 1 M H2O2 and 0.5 mM iron(III), PFOA was degraded by 89% within 150 min. Hydroxyl radical does not react with PFOA, but systems producing only superoxide promoted 68% PFOA degradation within 150 min. In systems producing only hydroperoxide, the level of PFOA degradation was 80% over 150 min. The generation of near-stoichiometric equivalents of fluoride during PFOA degradation and the lack of detectable degradation products suggest PFOA may be mineralized by CHP. CHP process conditions can be adjusted during treatability studies to increase the flux of superoxide and hydroperoxide to treat ...

Manganese superoxide dismutase depletion in murine hematopoietic stem cells perturbs iron homeostasis, globin switching, and epigenetic control in erythrocyte precursorcells

Heme synthesis partially occurs in the mitochondrial matrix; thus there is a high probability that enzymes and intermediates important in the production of heme will be exposed to metabolic by-products including reactive oxygen species. In addition, the need for ferrous iron for heme production, Fe/S coordination, and other processes occurring in the mitochondrial matrix suggests that aberrant fluxes of reactive oxygen species in this compartment might perturb normal iron homeostasis. Manganese superoxide dismutase (Sod2) is an antioxidant enzyme that governs steady-state levels of the superoxide in the mitochondrial matrix. Using hematopoietic stem cell-specific conditional Sod2 knockout mice we observed increased superoxide concentrations in red cell progeny, which caused significant pathologies including impaired erythrocytes and decreased ferrochelatase activity. Animals lacking Sod2 expression in erythroid precursors also displayed extramedullary hematopoiesis and systemic iron redistribution. Additionally, the increase in superoxide flux in erythroid precursors caused abnormal gene regulation of hematopoietic transcription factors, globins, and iron-response genes. Moreover, the erythroid precursors also displayed evidence of global changes in histone posttranslational modifications, a likely cause of at least some of the aberrant gene expression noted. From a therapeutic translational perspective, mitochondrially targeted superoxide-scavenging antioxidants partially rescued the observed phenotype. Taken together, our findings illuminate the superoxide sensitivity of normal iron homeostasis in erythrocyte precursors and suggest a probable link between mitochondrial redox metabolism and epigenetic control of nuclear gene regulation during mammalian erythropoiesis.

Disruption of Mitochondrial Superoxide Flux Causes Abnormal Heme Synthesis and Hemoglobin Gene Regulation in Erythroid Precursor Cells

Disruption of Mitochondrial Superoxide Flux Causes Abnormal Heme Synthesis and Hemoglobin Gene Regulation in Erythroid Precursor Cells

Real-time monitoring of superoxide accumulation and antioxidant activity in a brain slice model using an electrochemical cytochrome c biosensor

The overproduction of reactive oxygen species and the resulting damage are central to the pathology of many diseases. The study of the temporal and spatial accumulation of reactive oxygen species has been limited because of the lack of specific probes and techniques capable of continuous measurement. We demonstrate the use of a miniaturized electrochemical cytochrome c (Cyt c) biosensor for real-time measurements and quantitative assessment of superoxide production and inactivation by natural and engineered antioxidants in acutely prepared brain slices from mice. Under control conditions, superoxide radicals produced from the hippocampal region of the brain in 400-μm-thick sections were well within the range of detection of the electrode. Exposure of the slices to ischemic conditions increased the superoxide production twofold and measurements from the slices were stable over a 3- to 4-h period. The stilbene derivative and anion channel inhibitor 4,4′-diisothiocyano-2,2′-disulfonic stilbene markedly reduced the extracellular superoxide signal under control conditions, suggesting that a transmembrane flux of superoxide into the extracellular space may occur as part of normal redox signaling. The specificity of the electrode for superoxide released by cells in the hippocampus was verified by the exogenous addition of superoxide dismutase (SOD), which decreased the superoxide signal in a dose-dependent manner. Similar results were seen with the addition of the SOD mimetic cerium oxide nanoparticles (nanoceria), in that the superoxide anion radical scavenging activity of nanoceria with an average diameter of 15 nm was equivalent to 527 U of SOD for each 1 μg/ml of nanoceria added. This study demonstrates the potential of electrochemical biosensors for studying real-time dynamics of reactive oxygen species in a biological model and the utility of these measurements in defining the relative contribution of superoxide to oxidative injury.

Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death

Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both autophagic cell death and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (−3, −7, and −9). Substantial depolarization of mitochondria occurred after 1h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.

Mitochondrial Thiols in Antioxidant Protection and Redox Signaling: Distinct Roles for Glutathionylation and Other Thiol Modifications

The mitochondrial matrix contains much of the machinery at the heart of metabolism. This compartment is also exposed to a high and continual flux of superoxide, hydrogen peroxide, and related reactive species. To protect mitochondria from these sources of oxidative damage, there is an integrated set of thiol systems within the matrix comprising the thioredoxin/peroxiredoxin/methionine sulfoxide reductase pathways and the glutathione/glutathione peroxidase/glutathione-S-transferase/glutaredoxin pathways that in conjunction with protein thiols prevent much of this oxidative damage. In addition, the changes in the redox state of many components of these mitochondrial thiol systems may transduce and relay redox signals within and through the mitochondrial matrix to modulate the activity of biochemical processes. Here, mitochondrial thiol systems are reviewed, and areas of uncertainty are pointed out, focusing on recent developments in our understanding of their roles. The areas of particular focus are on the multiple, overlapping roles of mitochondrial thiols and on understanding how these thiols contribute to both antioxidant defenses and redox signaling. Recent technical progress in the identification and quantification of thiol modifications by redox proteomics means that many of the questions raised about the multiple roles of mitochondrial thiols can now be addressed.

Flavin-Linked Erv-Family Sulfhydryl Oxidases Release Superoxide Anion during Catalytic Turnover

Typically, simple flavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen peroxide without release of significant levels of the superoxide ion. However, two evolutionarily related single-domain sulfhydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predominantly found in the mitochondrial intermembrane) release up to ~30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofactor within the helix-rich Erv domain. Subsequent reduction of the flavin is followed by transfer of reducing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (THP) as an alternative reducing substrate to dithiothreitol (DTT). THP, and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode, but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the formation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP), respectively. This superoxide-dependent amplification of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemiluminescence method using a luciferin analogue (MCLA) when 2 mM DTT was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~65% when monomeric mutants of the normally homodimeric enzyme were used. In contrast, monomeric multidomain quiescin sulfhydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed.

Simulation of generation–collection experiments with homogeneous kinetics: application to electrochemical investigation of superoxide radical anion generation by osteoclasts on bone

We report simulations of electrochemical generation-collection experiments in which the generator is a small disc producing a specified time-dependent flux of the analyte and the collector is a large planar electrode which collects the analyte at the mass transport-controlled rate. This geometry corresponds to many experiments in bioelectrochemistry where a relatively large sensor is used to detect the products of a cell's metabolism at low concentration. In particular, our simulations are motivated by attempts to understand our results on the detection of the superoxide radical anion burst generated by osteoclasts (bone-resorbing cells) in response to various stimuli. Superoxide is present at low levels and disproportionates in aqueous media; however, the homogeneous kinetics are included in our simulations and the results show that it is possible to estimate the magnitude of the flux of superoxide produced by the cells and to accurately determine the time-dependence of the flux in response to stimuli such as injection of parathyroid hormone, vitamin D(3) and pertussis toxin. In all these cases, the superoxide anion flux was successfully modeled as uniform across the cell surface with time-dependence of the form j(0)e(-k(d)t) + j(∞). j(∞) is the sustained flux of superoxide and the first-order rate constant k(d) and the magnitude j(0) describe the transient component of the flux. The simulations indicate that for cell-electrode gaps D approximately < √(D/k(d)), where D is the diffusion coefficient, the value of k(d) can be accurately extracted from the time-dependence of the collector current without detailed knowledge of parameters which are hard to measure during the experiment, e.g., the cell radius a and cell-electrode separation d. In the case of parathyroid hormone, the first-order rate constant describing the decay of the transient component was k(d) = 1.8 ± 0.8 × 10(-1) s(-1), but much slower decays were observed in response to pertussis toxin (k(d) = 1.5 ± 0.5 × 10(-2) s(-1)) and vitamin D(3) (k(d) = 1.1 ± 0.5 × 10(-3) s(-1)).

Chemical model systems for cellular nitros(yl)ation reactions

S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ( NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1−)) as an essential intermediate. This requires low cellular fluxes of NO and superoxide ( O 2 −), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine, equal fluxes of NO and O 2 − were generated and modulated by the addition of NO donors or Cu,Zn-superoxide dismutase. As reactants for S-nitros(yl)ation, NADP +-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of NO and O 2 − at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and N-nitrosation) demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of NO was as efficient as the 3 NO/1 O 2 − system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N 2O 3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described NO/ O 2 − flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.

Important role of peroxynitrite‐nitric oxide interaction for S‐, N‐ and C‐nitrosation reactions

Biological nitrosations belong to a set of redox based chemical posttranslational modifications of proteins that are involved in cellular regulation. Peroxynitrite is a central player in redox signaling and is expected to play a role in nitrosation. In earlier work we and others have suggested that nitrosation is mediated by intermediates that arise by cellular fluxes of nitric oxide and superoxide but the nature of such intermediates remained elusive. In the present study we have investigated model systems for cellular nitrosation using phenol, diaminonaphthaline and isocitrate dehydrogenase as substrates for C‐, N‐, and S‐nitrosation. The thermal aerobic decay of Sin‐1 into equal amounts of NO and superoxide was used and NONOate was added as a NO source which lead to a nitrosation maximum at a NO:superoxide ratio of 3:1. When Cu, Zn‐SOD was present during the decay of Sin‐1 a gradual shift to NO formation was indicated by use of an NO‐sensitive electrode and a SOD dependent maximum of nitrosations was obtained also in this system. Qualitatively similar results were obtained when the SOD mimetic osmium tetroxide was used. We conclude from the various systems investigated that biological nitrosations can arise from fluxes of NO and superoxide at ratios of about 3:1. It is too early to speculate on the mechanism involved but the systems presented seem to be valid models to elucidate biological nitrosation reactions.