Natalia Rios

Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34

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 × 10(5) 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 × 10(6) 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 × 10(5) 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 Mn(III) to yield Mn(IV) 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.

Sensitive Detection and Estimation of Cell-Derived Peroxynitrite Fluxes Using Fluorescein-Boronate

The specific and sensitive detection of peroxynitrite (ONOO-/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (∼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×106M-1s-1, a million times faster than the rate constant measured for H2O2 (k=1.7M-1s-1) and 2,700 faster than HOCl (6.2×102M-1s-1) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1μMs-1, while immunostimulated macrophages do so in the order of ∼1μMs-1 inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1mM for 24h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.

Nitric oxide diffusion to red blood cells limits extracellular, but not intraphagosomal, peroxynitrite formation by macrophages

Macrophage-derived nitric oxide ((•)NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by (•)NO itself or (•)NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O(2)(•-)). In vivo, the facile extracellular diffusion of (•)NO as well as different competing consumption routes limit its bioavailability for the reaction with O(2)(•-) and, hence, peroxynitrite formation. In this work, we evaluated the extent by which (•)NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O(2)(•-) and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of (•)NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of (•)NO and O(2)(•-)-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of (•)NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O(2)(•-) reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of (•)NO and O(2)(•-)-forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, (•)NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.

Nitro-Arachidonic Acid Prevents Angiotensin II-Induced Mitochondrial Dysfunction in a Cell Line of Kidney Proximal Tubular Cells

Nitro-arachidonic acid (NO2-AA) is a cell signaling nitroalkene that exerts anti-inflammatory activities during macrophage activation. While angiotensin II (ANG II) produces an increase in reactive oxygen species (ROS) production and mitochondrial dysfunction in renal tubular cells, little is known regarding the potential protective effects of NO2-AA in ANG II-mediated kidney injury. As such, this study examines the impact of NO2-AA on ANG II-induced mitochondrial dysfunction in an immortalized renal proximal tubule cell line (HK-2 cells). Treatment of HK-2 cells with ANG II increases the production of superoxide (O2●-), nitric oxide (●NO), inducible nitric oxide synthase (NOS2) expression, peroxynitrite (ONOO-) and mitochondrial dysfunction. Using high-resolution respirometry, it was observed that the presence of NO2-AA prevented ANG II-mediated mitochondrial dysfunction. Attempting to address mechanism, we treated isolated rat kidney mitochondria with ONOO-, a key mediator of ANG II-induced mitochondrial damage, in the presence or absence of NO2-AA. Whereas the activity of succinate dehydrogenase (SDH) and ATP synthase (ATPase) were diminished upon exposure to ONOO-, they were restored by pre-incubating the mitochondria with NO2-AA. Moreover, NO2-AA prevents oxidation and nitration of mitochondrial proteins. Combined, these data demonstrate that ANG II-mediated oxidative damage and mitochondrial dysfunction is abrogated by NO2-AA, identifying this compound as a promising pharmacological tool to prevent ANG II–induced renal disease.

Fluorescence and chemiluminescence approaches for peroxynitrite detection

ABSTRACT In the last two decades, there has been a significant advance in understanding the biochemistry of peroxynitrite, an endogenously‐produced oxidant and nucleophile. Its relevance as a mediator in several pathologic states and the aging process together with its transient character and low steady‐state concentration, motivated the development of a variety of techniques for its unambiguous detection and estimation. Among these, fluorescence and chemiluminescence approaches have represented important tools with enhanced sensitivity but usual limited specificity. In this review, we analyze selected examples of molecular probes that permit the detection of peroxynitrite by fluorescence and chemiluminescence, disclosing their mechanism of reaction with either peroxynitrite or peroxynitrite‐derived radicals. Indeed, probes have been divided into 1) redox probes that yield products by a free radical mechanism, and 2) electrophilic probes that evolve to products secondary to the nucleophilic attack by peroxynitrite. Overall, boronate‐based compounds are emerging as preferred probes for the sensitive and specific detection and quantitation. Moreover, novel strategies involving genetically‐modified fluorescent proteins with the incorporation of unnatural amino acids have been recently described as peroxynitrite sensors. This review analyzes the most commonly used fluorescence and chemiluminescence approaches for peroxynitrite detection and provides some guidelines for appropriate experimental design and data interpretation, including how to estimate peroxynitrite formation rates in cells. Graphical abstract Figure. No Caption available. HighlightsPeroxynitrite can be detected by fluorescent and chemiluminescent probes.Probes can react with peroxynitrite by redox‐ or nucleophilic‐based mechanisms.Current approaches allow peroxynitrite visualization in cells and tissues.Boronate‐based chemical probes are highly selective for peroxynitrite detection.Genetically‐encoded probes with reactive unnatural amino acids are being developed.

Iron‐sulfur glutaredoxin 2 protects oligodendrocytes against damage induced by nitric oxide release from activated microglia

Demyelinated brain lesions, a hallmark of autoimmune neuroinflammatory diseases like multiple sclerosis, result from oligodendroglial cell damage. Activated microglia are considered a major source of nitric oxide and subsequent peroxynitrite‐mediated damage of myelin. Here, we provide biochemical and biophysical evidence that the oxidoreductase glutaredoxin 2 inhibits peroxynitrite formation by transforming nitric oxide into dinitrosyl‐diglutathionyl‐iron‐complexes. Glutaredoxin 2 levels influence both survival rates of primary oligodendrocyte progenitor cells and preservation of myelin structure in cerebellar organotypic slice cultures challenged with activated microglia or nitric oxide donors. Of note, glutaredoxin 2‐mediated protection is not linked to its enzymatic activity as oxidoreductase, but to the disassembly of its uniquely coordinated iron‐sulfur cluster using glutathione as non‐protein ligand. The protective effect of glutaredoxin 2 is connected to decreased protein carbonylation and nitration. In line, brain lesions of mice suffering from experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, show decreased glutaredoxin 2 expression and increased nitrotyrosine formation indicating that this type of protection is missing in the inflamed central nervous system. Our findings link inorganic biochemistry to neuroinflammation and identify glutaredoxin 2 as a protective factor against neuroinflammation‐mediated myelin damage. Thus, improved availability of glutathione‐coordinated iron‐sulfur clusters emerges as a potential therapeutic approach in inflammatory demyelination.

Peroxynitrite Formation and Detection in Living Cells

Abstract Different cell types produce peroxynitrite under conditions of simultaneous generation of its precursor radicals, superoxide and nitric oxide. The detection of cell-derived peroxynitrite is technically challenging due to its short biological half-life and low steady-state concentration and the shortage of specific methods. However, appropriate use of molecular footprints and probes together with a wise application of pharmacological and genetic approaches, allow for its unambiguous detection. Among the molecular footprints left by peroxynitrite, the measurement and identification of tyrosine-nitrated proteins is of prime relevance. Regarding molecular probes, recent advances on the characterization of the reactivity of boronate-based molecules with peroxynitrite have opened new and more specific ways for cellular detection. A critical analysis of the chemical basis and usefulness of the existing methods utilized for the cellular detection of peroxynitrite will be performed. Also, the role of intracellular modulators of peroxynitrite reactivity and levels (e.g., CO2, uric acid, peroxiredoxins) and how they can influence the detected levels will be assessed. The accurate cellular detection of peroxynitrite in different cellular and extracellular compartments and the estimation of its formation rates, represent fundamental steps to understand how nitric oxide-derived oxidants affect biological processes, including mitochondrial dysfunction and cell death.

Correction: Nitro-Arachidonic Acid Prevents Angiotensin II Induced Mitochondrial Dysfunction in Kidney Proximal Tubular Cells.

[This corrects the article DOI: 10.1371/journal.pone.0150459.].