DNIC can be formed in aqueous media in the absence of thiols via mechanisms that depend exclusive... more DNIC can be formed in aqueous media in the absence of thiols via mechanisms that depend exclusively on Fe(II) and NO. However, these reactions do not take place at intracellular concentrations of Fe(II) and NO, reinforcing the relevance of thiols to assist Fe(II) to Fe(I) reduction during DNIC formation in biological media.
Copper nitrite reductase mimetics were shown to have their catalysis influenced by the secondary ... more Copper nitrite reductase mimetics were shown to have their catalysis influenced by the secondary coordination sphere and hydrogen bonding.
This work evaluated the analgesic and anti-inflammatory activity of ruthenium(II) complexes trans... more This work evaluated the analgesic and anti-inflammatory activity of ruthenium(II) complexes trans-[Ru(NO(+))(NH3)4(L)](BF4)3 and [Ru(NH3)5(L)](BF4)3 containing the nonsteroidal anti-inflammatory drugs nicotinic acid (Hnic) and its isomer isonicotinic acid (ina) as ligands (L). The anti-nociceptive potential of these complexes and the free ligands (noncoordinated to ruthenium) was tested in different models with doses ranging from 1 to 100 μmol/kg. The ligands themselves were inactive; however, the ruthenium complexes containing Hnic and ina inhibited mechanical hyperalgesia induced by prostaglandin E2, carrageenan-induced hyperalgesia, and antigen-induced arthritis. Moreover, the ruthenium complexes inhibited overt nociception induced by formalin, acetic acid, capsaicin, and cinnamaldehyde. The mechanism involved in the anti-nociceptive effects of the ruthenium complexes suggested that ATP-sensitive K(+) channel pathways were not involved because glibenclamide did not affect their a...
Protein S-nitrosation is an important consequence of NO●·metabolism with implications in physiolo... more Protein S-nitrosation is an important consequence of NO●·metabolism with implications in physiology and pathology. The mechanisms responsible for S-nitrosation in vivo remain debatable and kinetic data on protein S-nitrosation by different agents are limited. 2-Cys peroxiredoxins, in particular Prx1 and Prx2, were detected as being S-nitrosated in multiple mammalian cells under a variety of conditions. Here, we investigated the kinetics of Prx1 S-nitrosation by nitrosoglutathione (GSNO), a recognized biological nitrosating agent, and by the dinitrosyl-iron complex of glutathione (DNIC-GS; [Fe(NO)2(GS)2]−), a hypothetical nitrosating agent. Kinetics studies following the intrinsic fluorescence of Prx1 and its mutants (C83SC173S and C52S) were complemented by product analysis; all experiments were performed at pH 7.4 and 25 ℃. The results show GSNO-mediated nitrosation of Prx1 peroxidatic residue ( k + N O C y s 52 = 15.4 ± 0.4 M−1. s−1) and of Prx1 Cys83 residue ( k + N O C y s 83 = ...
Thiyl radicals are detected by EPR as co-products of dinitrosyl iron complex (DNIC) formation. In... more 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...
The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the hig... more The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive hydroxyl radical, which forms through a redox reaction with hydrogen peroxide. Peroxynitrite is a biologically relevant peroxide produced by the recombination of nitric oxide and superoxide. It is a strong oxidant that may be involved in multiple pathological conditions, but whether and how it interacts with the LIP are unclear. Here, using fluorescence spectroscopy, we investigated the interaction between the LIP and peroxynitrite by monitoring peroxynitrite-dependent accumulation of nitrosated and oxidized fluorescent intracellular indicators. We found that, in murine macrophages, removal of the LIP with membrane-permeable iron chelators sustainably accelerates the peroxynitrite-dependent oxidation and nitrosation of these indicators. These observations could not be reproduced in cell-free assays, indicating that the chelator-enhancing effect on peroxynitrite-dependent mod...
Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs ... more Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cysand Cys) by hydrogen peroxide (= 17.3 ± 1.3 msat pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (= (6.9 ± 0.2) × 10ms), and both Cys re...
Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme-peroxidases. ... more Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme-peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is plenty amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable to hydrogen peroxide. The second-order rate constants of these reactions were 4.9 x 10(5) M(-1)s(-1) and 2.3 x 10(6) M(-1) s(-1) for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme, then, it oxidizes the peroxidatic cysteine and, finally, the rate-limiting disulfide bond is formed. Of...
DNIC can be formed in aqueous media in the absence of thiols via mechanisms that depend exclusive... more DNIC can be formed in aqueous media in the absence of thiols via mechanisms that depend exclusively on Fe(II) and NO. However, these reactions do not take place at intracellular concentrations of Fe(II) and NO, reinforcing the relevance of thiols to assist Fe(II) to Fe(I) reduction during DNIC formation in biological media.
Copper nitrite reductase mimetics were shown to have their catalysis influenced by the secondary ... more Copper nitrite reductase mimetics were shown to have their catalysis influenced by the secondary coordination sphere and hydrogen bonding.
This work evaluated the analgesic and anti-inflammatory activity of ruthenium(II) complexes trans... more This work evaluated the analgesic and anti-inflammatory activity of ruthenium(II) complexes trans-[Ru(NO(+))(NH3)4(L)](BF4)3 and [Ru(NH3)5(L)](BF4)3 containing the nonsteroidal anti-inflammatory drugs nicotinic acid (Hnic) and its isomer isonicotinic acid (ina) as ligands (L). The anti-nociceptive potential of these complexes and the free ligands (noncoordinated to ruthenium) was tested in different models with doses ranging from 1 to 100 μmol/kg. The ligands themselves were inactive; however, the ruthenium complexes containing Hnic and ina inhibited mechanical hyperalgesia induced by prostaglandin E2, carrageenan-induced hyperalgesia, and antigen-induced arthritis. Moreover, the ruthenium complexes inhibited overt nociception induced by formalin, acetic acid, capsaicin, and cinnamaldehyde. The mechanism involved in the anti-nociceptive effects of the ruthenium complexes suggested that ATP-sensitive K(+) channel pathways were not involved because glibenclamide did not affect their a...
Protein S-nitrosation is an important consequence of NO●·metabolism with implications in physiolo... more Protein S-nitrosation is an important consequence of NO●·metabolism with implications in physiology and pathology. The mechanisms responsible for S-nitrosation in vivo remain debatable and kinetic data on protein S-nitrosation by different agents are limited. 2-Cys peroxiredoxins, in particular Prx1 and Prx2, were detected as being S-nitrosated in multiple mammalian cells under a variety of conditions. Here, we investigated the kinetics of Prx1 S-nitrosation by nitrosoglutathione (GSNO), a recognized biological nitrosating agent, and by the dinitrosyl-iron complex of glutathione (DNIC-GS; [Fe(NO)2(GS)2]−), a hypothetical nitrosating agent. Kinetics studies following the intrinsic fluorescence of Prx1 and its mutants (C83SC173S and C52S) were complemented by product analysis; all experiments were performed at pH 7.4 and 25 ℃. The results show GSNO-mediated nitrosation of Prx1 peroxidatic residue ( k + N O C y s 52 = 15.4 ± 0.4 M−1. s−1) and of Prx1 Cys83 residue ( k + N O C y s 83 = ...
Thiyl radicals are detected by EPR as co-products of dinitrosyl iron complex (DNIC) formation. In... more 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...
The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the hig... more The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive hydroxyl radical, which forms through a redox reaction with hydrogen peroxide. Peroxynitrite is a biologically relevant peroxide produced by the recombination of nitric oxide and superoxide. It is a strong oxidant that may be involved in multiple pathological conditions, but whether and how it interacts with the LIP are unclear. Here, using fluorescence spectroscopy, we investigated the interaction between the LIP and peroxynitrite by monitoring peroxynitrite-dependent accumulation of nitrosated and oxidized fluorescent intracellular indicators. We found that, in murine macrophages, removal of the LIP with membrane-permeable iron chelators sustainably accelerates the peroxynitrite-dependent oxidation and nitrosation of these indicators. These observations could not be reproduced in cell-free assays, indicating that the chelator-enhancing effect on peroxynitrite-dependent mod...
Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs ... more Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cysand Cys) by hydrogen peroxide (= 17.3 ± 1.3 msat pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (= (6.9 ± 0.2) × 10ms), and both Cys re...
Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme-peroxidases. ... more Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme-peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is plenty amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable to hydrogen peroxide. The second-order rate constants of these reactions were 4.9 x 10(5) M(-1)s(-1) and 2.3 x 10(6) M(-1) s(-1) for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme, then, it oxidizes the peroxidatic cysteine and, finally, the rate-limiting disulfide bond is formed. Of...
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