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2. Dynamics of Dinitrosyl Iron Complex (DNIC) Formation with Low Molecular Weight Thiols

Author

Truzzi, Daniela R, Augusto, Ohara, Iretskii, Alexei V, and Ford, Peter C

Subjects
Chemical Sciences, Physical Chemistry, Inorganic Chemistry, Physical Chemistry (incl. Structural), Other Chemical Sciences, Inorganic & Nuclear Chemistry, Inorganic chemistry, Macromolecular and materials chemistry
Abstract

Dinitrosyl iron complexes (DNICs) are ubiquitous in mammalian cells and tissues producing nitric oxide (NO) and have been argued to play key physiological and pathological roles. Nonetheless, the mechanism and dynamics of DNIC formation in aqueous media remain only partially understood. Here, we report a stopped-flow kinetics and density functional theory (DFT) investigation of the reaction of NO with ferrous ions and the low molecular weight thiols glutathione (GSH) and cysteine (CysSH) as well as the peptides WCGPC and WCGPY to produce DNICs in pH 7.4 aqueous media. With each thiol, a two-stage reaction pattern is observed. The first stage involves several rapidly established pre-equilibria leading to a ferrous intermediate concluded to have the composition FeII(NO)(RS)2(H2O)x (C). In the second stage, C undergoes rate-limiting, unimolecular autoreduction to give thiyl radical (RS•) plus the mononitrosyl Fe(I) complex FeI(NO)(RS)(H2O)x following the reactivity order of CysSH > WCGPC > WCGPY > GSH. Time course simulations using the experimentally determined kinetics parameters demonstrate that, at a NO flux characteristic of inflammation, DNICs will be rapidly formed from intracellular levels of ferrous iron and thiols. Furthermore, the proposed mechanism offers a novel pathway for S-nitroso thiol (RSNO) formation in a biological environment.

Published
2019

3. Thiyl radicals are co-products of dinitrosyl iron complex (DNIC) formation

Author

Truzzi, Daniela R, Augusto, Ohara, and Ford, Peter C

Subjects
Inorganic Chemistry, Chemical Sciences, Organic Chemistry, Chemical sciences, Engineering
Abstract

Thiyl radicals are detected by EPR as co-products of dinitrosyl iron complex (DNIC) formation. In demonstrating that DNIC formation generates RS˙ in a NO rich environment, these results provide a novel route for S-nitroso thiol formation.

Published
2019

5. Production of Peroxymonocarbonate by Steady-State Micromolar H2O2and Activated Macrophages in the Presence of CO2/HCO3–Evidenced by Boronate Probes

Author

Linares, Edlaine, Severino, Divinomar, Truzzi, Daniela R., Rios, Natalia, Radi, Rafael, and Augusto, Ohara

Abstract

Peroxymonocarbonate (HCO4–/HOOCO2–) is produced by the reversible reaction of CO2/HCO3–with H2O2(K= 0.33 M–1, pH 7.0). Although produced in low yields at physiological pHs and H2O2and CO2/HCO3–concentrations, HCO4–oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of H2O2. Boronate probes are known examples because HCO4–reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of H2O2and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO4–formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state H2O2concentrations (2–18 μM) in Pi buffer at pH 7.4 and 37 °C. Then, CBE (100 μM) was added and fluorescence increase was monitored with time. The results showed that each steady-state H2O2concentration reacted more rapidly (∼30%) in the presence of CO2/HCO3–(25 mM) than in its absence, and the data permitted the calculation of consistent rate constants. Also, RAW 264.7 macrophages were activated with phorbol 12-myristate 13-acetate (PMA) (1 μg/mL) at pH 7.4 and 37 °C to produce a time-dependent H2O2concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO3–equilibrated with 0, 5, or 10% CO2, respectively. In the presence of CBE or Fl-B (30 μM), a time-dependent increase in the fluorescence of the bulk solution was observed, which was higher in the presence of CO2/HCO3–in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO4–and boronate probes can evidence and distinguish it from H2O2under biologically relevant concentrations of H2O2and CO2/HCO3–.

Published
2024
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9. Even free radicals should follow some rules: A Guide to free radical research terminology and methodology

Author

Forman, Henry Jay, Augusto, Ohara, Brigelius-Flohe, Regina, Dennery, Phyllis A, Kalyanaraman, Balaraman, Ischiropoulos, Harry, Mann, Giovanni E, Radi, Rafael, Roberts, L Jackson, Vina, Jose, and Davies, Kelvin JA

Subjects
Animals, Antioxidants, Fluorescent Dyes, Free Radical Scavengers, Free Radicals, Humans, Lipid Peroxidation, Reactive Nitrogen Species, Reactive Oxygen Species, Terminology as Topic, Thiobarbituric Acid Reactive Substances, Fluorescent dye, Reactive oxygen species, Reactive nitrogen species, Thiobarbituric acid, F2-isoprostane, Antioxidant, alpha-Tocopherol, Radical scavenging, Free radicals, α-Tocopherol, Medicinal and Biomolecular Chemistry, Biochemistry and Cell Biology, Medical Biochemistry and Metabolomics, Biochemistry & Molecular Biology
Abstract

Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species.

Published
2015

19. Oxidative Modification of Proteins: From Damage to Catalysis, Signaling, and Beyond

Author

Demasi, Marilene, primary, Augusto, Ohara, additional, Bechara, Etelvino J.H., additional, Bicev, Renata N., additional, Cerqueira, Fernanda M., additional, da Cunha, Fernanda M., additional, Denicola, Ana, additional, Gomes, Fernando, additional, Miyamoto, Sayuri, additional, Netto, Luis E.S., additional, Randall, Lía M., additional, Stevani, Cassius V., additional, and Thomson, Leonor, additional

Published
2021
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37. Presence of Ser instead of Thr in the Catalytic Triad of Typical 2-Cys Prx Increases their Resistance to Hyperoxidation and Inactivation

Author

Santos, Melina, primary, Breyer, Carlos, additional, Tairum, Carlos, additional, Pires de Oliveira, Ana Laura, additional, Montanhero, Vitória Isabela, additional, Toledo-Silva, Guilherme, additional, Mori, Gustavo Maruyama, additional, Toyama, Marcos Hikari, additional, Augusto, Ohara, additional, Netto, Luis, additional, and Oliveira, Marcos, additional

Published
2020
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45. EPR detection of glutathiyl and hemoglobin-cysteinyl radicals during the interaction of peroxynitrite with human erythrocytes

Author

Augusto, Ohara, Menezes, Silvia Lopes de, Linares, Edlaine, Romero, Natalia, Radi, Rafael, and Denicola, Ana

Subjects
Biochemistry -- Research, Erythrocytes -- Physiological aspects, Hemoglobin -- Physiological aspects, Electron paramagnetic resonance -- Usage, Radicals (Chemistry) -- Physiological aspects, Nitrites -- Physiological aspects, Biological sciences, Chemistry
Abstract

Research has been conducted on peroxynitrite. The hypothesis that free radicals can be produced from this peroxynitrite has been tested, and the use of electron paramagnetic resonance spin trapping methodology in investigating interactions of peroxynitrite with human erythrocytes is described.

Published
2002

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