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Peroxymonocarbonate (HCO 4 - /HOOCO 2 - ) is produced by the reversible reaction of CO 2 /HCO 3 - with H 2 O 2 ( K = 0.33 M -1 , pH 7.0). Although produced in low yields at physiological pHs and H 2 O 2 and CO 2 /HCO 3 - concentrations, HCO 4 - oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of H 2 O 2 . Boronate probes are known examples because HCO 4 - reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of H 2 O 2 and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO 4 - formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state H 2 O 2 concentrations (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 H 2 O 2 concentration reacted more rapidly (∼30%) in the presence of CO 2 /HCO 3 - (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 H 2 O 2 concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO 3 - equilibrated with 0, 5, or 10% CO 2 , 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 CO 2 /HCO 3 - in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO 4 - and boronate probes can evidence and distinguish it from H 2 O 2 under biologically relevant concentrations of H 2 O 2 and CO 2 /HCO 3 - .

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 synthesized using three new tridentate ligands sharing the same N , N , N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n -butyl and n -propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ 2 O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κ O mode, and 15 N NMR experiments of a Zn 2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n -propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

High carbon dioxide tensions (hypercapnia) are toxic to mammals by both pH-dependent and pH-independent mechanisms that remain partially understood. Relevantly, carbon dioxide reacts with biologically ubiquitous oxygen metabolites such as peroxynitrite and hydrogen peroxide to produce carbonate radical and peroxymonocarbonate, respectively. These metabolites are redox active making it timely to discuss the potential role of carbon dioxide redox metabolites in oxidative eustress and oxidative distress conditions., Competing Interests: Conflict of interestThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2021.)

Dinitrosyl iron complexes (DNICs) are spontaneously and rapidly generated in cells. Their assembly requires nitric oxide (NO), biothiols, and nonheme iron, either labile iron or iron-sulfur clusters. Despite ubiquitous detection by electron paramagnetic resonance in NO-producing cells, the DNIC's chemical biology remains only partially understood. In this Forum Article, we address the reaction mechanisms for endogenous DNIC formation, with a focus on a labile iron pool as the iron source. The capability of DNICs to promote S-nitrosation is discussed in terms of S -nitrosothiol generation associated with the formation and chemical reactivity of DNICs. We also highlight how elucidation of the chemical reactivity and the dynamics of DNICs combined with the development of detection/quantification methods can provide further information regarding their participation in physiological and pathological processes.

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 Cys 83 residue ( k + N O C y s 83 = 1.7 ± 0.4 M -1 . s -1 ). The reaction of nitrosated Prx1 with GSH was also monitored and provided a second-order rate constant for Prx1Cys 52 NO denitrosation of k - N O C y s 52 = 14.4 ± 0.3 M -1 . s -1 . In contrast, the reaction of DNIC-GS with Prx1 did not nitrosate the enzyme but formed DNIC-Prx1 complexes. The peroxidatic Prx1 Cys was identified as the residue that more rapidly replaces the GS ligand from DNIC-GS ( k D N I C C y s 52 = 7.0 ± 0.4 M -1 . s -1 ) to produce DNIC-Prx1 ([Fe(NO) 2 (GS)(Cys 52 -Prx1)] - ). Altogether, the data showed that in addition to S-nitrosation, the Prx1 peroxidatic residue can replace the GS ligand from DNIC-GS, forming stable DNIC-Prx1, and both modifications disrupt important redox switches.

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 Fe II (NO)(RS) 2 (H 2 O) x ( C ). In the second stage, C undergoes rate-limiting, unimolecular autoreduction to give thiyl radical (RS • ) plus the mononitrosyl Fe(I) complex Fe I (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.

2-Cys peroxiredoxins (Prxs) rapidly reduce H 2 O 2 , thereby acting as antioxidants and also as sensors and transmitters of H 2 O 2 signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high H 2 O 2 levels. Under these conditions, H 2 O 2 oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (C P SOH) to the sulfinate (C P SO 2 - ) and sulfonated (C P SO 3 - ) forms, redirecting the C P SOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the C P SOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to H 2 O 2 and was selected here to investigate the effect of a physiological concentration of HCO 3 - /CO 2 (25 mm) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO 3 - /CO 2 increases Prx1 hyperoxidation and inactivation both in the presence of excess H 2 O 2 and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO 3 - /CO 2 was due to HCO 4 - , a peroxide present in equilibrated solutions of H 2 O 2 and HCO 3 - /CO 2 Indeed, additional experiments and calculations uncovered that HCO 4 - oxidizes C P SOH to C P SO 2 - with a second-order rate constant 2 orders of magnitude higher than that of H 2 O 2 ((1.5 ± 0.1) × 10 5 and (2.9 ± 0.2) × 10 3 m -1 ·s -1 , respectively) and that HCO 4 - is 250 times more efficient than H 2 O 2 at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO 3 - /CO 2 pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity., (© 2019 Truzzi et al.)

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.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

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 modifications of the indicators depended on cell constituents, presumably including LIP, that react with these chelators. Moreover, neither free nor ferrous-complexed chelators stimulated intracellular or extracellular oxidative and nitrosative chemistries. On the basis of these results, LIP appears to be a relevant and competitive cellular target of peroxynitrite or its derived oxidants, and thereby it reduces oxidative processes, an observation that may change the conventional notion that the LIP is simply a cellular source of pro-oxidant iron., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

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 (Cys 53 and Cys 397 ) by hydrogen peroxide ( k = 17.3 ± 1.3 m -1 s -1 at 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 ( k = (6.9 ± 0.2) × 10 4 m -1 s -1 ), and both Cys residues were kinetically indistinguishable. Limited proteolysis, kinetic simulations, and MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which reacted with the resolving thiols at the active sites to produce disulfides ( i.e. Cys 53 -Cys 56 and Cys 397 -Cys 400 ). A fraction of peroxynitrite, however, decayed to radicals that hydroxylated and nitrated other active-site residues (Trp 52 , Trp 396 , and Tyr 393 ). Excess peroxynitrite promoted further PDI oxidation, nitration, inactivation, and covalent oligomerization. We conclude that these PDI modifications may contribute to the pathogenic mechanism of several diseases associated with dysfunctional PDI., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

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 large 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 with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 10 5 and 2.3 × 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 relevance, the disulfide bond formation was much slower in Prx2 ( k 3 = 0.31 s -1 ) than Prx1 ( k 3 = 14.9 s -1 ). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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 anti-nociceptive activities. However, the anti-nociceptive effect appears to be a consequence of the reduction in neutrophil migration and inhibition of the protein kinase C pathway.

Nitric oxide plays an important role in various biological processes, such as neurotransmission, blood pressure control, immunological responses, and antioxidant action. The control of its local concentration, which is crucial for obtaining the desired effect, can be achieved with exogenous NO-carriers. Coordination compounds, in particular ruthenium(III) and (II) amines, are good NO-captors and -deliverers. The chemical and photochemical properties of several ruthenium amine complexes as NO-carriers in vitro and in vivo have been reviewed. These nitrosyl complexes can stimulate mice hippocampus slices, promote the lowering of blood pressure in several in vitro and in vivo models, and control Trypanosoma cruzi and Leishmania major infections, and they are also effective against tumor cells in different models of cancer. These complexes can be activated chemically or photochemically, and the observed biological effects can be attributed to the presence of NO in the compound. Their efficiencies are explained on the basis of the [Ru(II)NO(+)](3+)/[Ru(II)NO(0)](2+) reduction potential, the specific rate constant for NO liberation from the [RuNO](2+) moiety, and the quantum yield of NO release., (Copyright © 2011. Published by Elsevier Inc.)

The trans-[Ru(NO)(NH(3))(4)(P(OH)(3))]Cl(3) complex was synthesized by reacting [Ru(H(2)O)(NH(3))(5)](2+) with H(3)PO(3) and characterized by spectroscopic ((31)P-NMR, δ = 68 ppm) and spectrophotometric techniques (λ = 525 nm, ε = 20 L mol(-1) cm(-1); λ = 319 nm, ε = 773 L mol(-1) cm(-1); λ = 241 nm, ε = 1385 L mol(-1) cm(-1); ν(NO(+)) = 1879 cm(-1)). A pK(a) of 0.74 was determined from infrared measurements as a function of pH for the reaction: trans-[Ru(NO)(NH(3))(4)(P(OH)(3))](3+) + H(2)O ⇌ trans-[Ru(NO)(NH(3))(4)(P(O(-))(OH)(2))](2+) + H(3)O(+). According to (31)P-NMR, IR, UV-vis, cyclic voltammetry and ab initio calculation data, upon deprotonation, trans-[Ru(NO)(NH(3))(4)(P(OH)(3))](3+) yields the O-bonded linkage isomer trans- [Ru(NO)(NH(3))(4)(OP(OH)(2))](2+), then the trans-[Ru(NO)(NH(3))(4)(OP(H)(OH)(2))](3+) decays to give the final products H(3)PO(3) and trans-[Ru(NO)(NH(3))(4)(H(2)O)](3+). The dissociation of phosphorous acid from the [Ru(NO)(NH(3))(4)](3+) moiety is pH dependent (k(obs) = 2.1 × 10(-4) s(-1) at pH 3.0, 25 °C)., (This journal is © The Royal Society of Chemistry 2011)