Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture.

Hydrogen sulfide (H2S) is an endogenous signaling molecule which is important for cardiovascular health, but its mechanism of action remains poorly understood. Here, we report measurements of H2S as well as its oxidized metabolites, termed small oxoacids of sulfur (SOS = HSOH and HOSOH), in four human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. We use a methodology that targets small molecular weight sulfur species; mass spectrometric analysis allows for species quantification to report cellular concentrations based on an H2S calibration curve. The production of H2S and SOS is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). In all the primary lines measured, the distributions of these three species were HOSOH >H2S > HSOH, with much higher SOS than seen previously in non-vascular cell lines. H2S and SOS were effluxed from smooth muscle cells in higher concentrations than endothelial cells. Aortic smooth muscle cells were used to examine changes under hypoxic growth conditions. Hypoxia caused notable increases in HSOH and ROS, which we attribute to enhanced sulfide quinone oxidase activity that results in reverse electron transport.

Correction: Kumar, M.R., et al. Characterization of Polysulfides, Polysulfanes, and Other Unique Species in the Reaction between GSNO and H2S. Molecules 2019, 24, 3090.

The authors wish to make the following corrections to this paper [...].

Characterization of Polysulfides, Polysulfanes, and Other Unique Species in the Reaction between GSNO and H2S.

Glutathione-based products, GSnX, of the reaction of hydrogen sulfide, H2S, S-nitroso glutathione, and GSNO, at varied stoichiometries have been analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) and chemical trapping experiments. A wide variety of glutathione-based species with catenated sulfur chains have been identified including sulfanes (GSSnG), sulfides (GSSnH), and sulfenic acids (GSnOH); sulfinic (GSnO2H) and sulfonic (GSnO3H) acids are also seen in reactions exposed to air. The presence of each species of GSnX within the original reaction mixtures was confirmed using Single Ion Chromatograms (SICs), to demonstrate the separation on the LC column, and given approximate quantification by the peak area of the SIC. Further, confirmation for different GSnX families was obtained by trapping with species-specific reagents. Several unique GSnX families have been characterized, including bridging mixed di- and tetra-valent polysulfanes and internal trithionitrates (GSNHSnH) with polysulfane branches. Competitive trapping experiments suggest that the polysulfane chains are formed via the intermediacy of sulfenic acid species, GSSnOH. In the presence of radical trap vinylcyclopropane (VCP) the relative distributions of polysulfane speciation are relatively unaffected, suggesting that radical coupling is not a dominant pathway. Therefore, we suggest polysulfane catenation occurs via reaction of sulfides with sulfenic acids.

The reaction between GSNO and H2S: On the generation of NO, HNO and N2O.

Several recent reports suggest that HNO may be produced endogenously by reaction of H2S and S-nitrosoglutathione (GSNO). This hypothesis was tested using deoxymyoglobin (MbFeII) to trap the expected HNO released from the target reaction, which should generate the stable HNO adduct, HNO-Mb, under anaerobic conditions. Under numerous experimental conditions, the sole globin product was NO-Mb, as characterized by absorbance, EPR, and NMR spectroscopies. Analogous reactions of GSNO with other biological reductants such as ascorbic acid, dithiothreitol, glutathione, and dithionite also yielded NO-Mb as the sole globin product; however, whereas analogous reduction of GSNO using NaBH4 generates HNO-Mb in high yield. Quantitative GC/MS analyses of reactions of GS15NO with H2S showed that the main reaction product was 15NO, with 15N2 produced at a comparable level to 15N2O. Overall yield of N2O is unchanged by the presence of MbFeII, discounting the intermediacy of either NO or HNO in its formation. Taken together, these results argue against the generation of free HNO as a major pathway in the reactions of GSNO with H2S, and instead imply some as yet uncharacterized intermediates generate the nitrogenic gases.

Mechanistic Investigations of Photoinduced Oxygenation of Ru(II) Bis-bipyridyl Flavonolate Complexes.

We previously reported that a Ru-bound flavonolate model of flavonol dioxygenases, [RuII(bpy)2(3-hydroxyfla)][PF6], photochemically reacts with dioxygen in two different manners. Broad-band excitation generates mixtures of products characteristic of 1,3-addition of dioxygen across the central pyrone ring, as is observed in enzymatic reactions. However, low temperature excitation at wavelengths longer than 400 nm generates a unique Ru-bound 2-benzoatophenylglyoxylate product resulting from a 1,2-dioxetane intermediate. Herein, we investigate this reactivity in a series of Ru(II)bis-bipyridyl flavonolate complexes [RuII(bpy)2(3-hydroxyflaR)][PF6] (bpy = 2,2'-bipyridine; fla = flavonolate; R = p-OMe (1), p-Me (2), p-H (3), p-Cl (4)), and [RuII(bpy)2(5-hydroxyfla)][PF6] (5). The complexes' structures, photophysical and electrochemical properties, and photochemical reactivity with oxygen were investigated in detail. Two different reaction product mixtures, from 1,2- and 1,3-additions of dioxygen, are observed by illumination into distinct excitation/emission manifolds. By analogy to previous reports of excited state intramolecular proton transfer, the two manifolds are attributed to tautomeric diradicals that predict the observed reactivity patterns.

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase.

Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products ((14)N/(15)N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metal-mediated electron transfer mechanism rather than metal activation of the substrate's inherent base-catalyzed reactivity.

Characterization of Endogenous and Extruded H2S and Small Oxoacids of Sulfur (SOS) in Cell Cultures.

This report characterizes and quantifies endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in a panel of cell lines including human cancer (A375 melanoma cells, HeLa cervical cells) and noncancer (HEK293 embryonic kidney cells), as well as E. coli DH5α and S. cerevisiae S288C. The methodology used is a translation of well-studied nucleophilic and electrophilic traps for cysteine and oxidized cysteines residues to target small molecular weight sulfur species; mass spectrometric analysis allows for species quantification. The observed intracellular concentrations of H2S and SOS vary in different cell types, from nanomolar to femtomolar, typically with H2S > HOSOH > HOSH. We propose the term sulfome, a subset of the metabolome, describing the nonproteinaceous metabolites of H2S; the sulfomic index is as a measure of the S-oxide redox status, which gives a profile of endogenous sulfur at different oxidation states. An important observation is that H2S and SOS were found to be continuously extruded into surrounding media against a concentration gradient, implying an active efflux process. Small molecule inhibition of several H2S generating enzymes suggest that SOS are not derived solely from H2S oxidation. Even after successful inhibition of H2S production, cells maintain constant efflux and repopulate H2S and SOS over time. This work proves that these small sulfur oxoacids are generated in cells of all types, and their efflux implies that they play a role in cell signaling and possibly other vascular physiology attributed to H2S.

Reactions of HNO with heme proteins: new routes to HNO-heme complexes and insight into physiological effects.

The formation and interconversion of nitrogen oxides has been of interest in numerous contexts for decades. Early studies focused on gas-phase reactions, particularly with regard to industrial and atmospheric environments, and on nitrogen fixation. Additionally, investigation of the coordination chemistry of nitric oxide (NO) with hemoglobin dates back nearly a century. With the discovery in the early 1980s that NO is biosynthesized as a molecular signaling agent, the literature has been focused on the biological effects of nitrogen oxides, but the original concerns remain relevant. For instance, hemoglobin has long been known to react with nitrite, but this reductase activity has recently been considered to be important to produce NO under hypoxic conditions. The association of nitrosyl hydride (HNO; also commonly referred to as nitroxyl) with heme proteins can also produce NO by reductive nitrosylation. Furthermore, HNO is considered to be an intermediate in bacterial denitrification, but conclusive identification has been elusive. The authors of this article have approached the bioinorganic chemistry of HNO from different perspectives, which have converged because heme proteins are important biological targets of HNO.

Nitrosyl hydride (HNO) as an O2 analogue: long-lived HNO adducts of ferrous globins.

Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O(2), and we have previously reported that deoxymyoglobin traps free HNO to form a stable adduct. In this report, we demonstrate that oxygen-binding hemoglobins from human, soy, and clam also trap HNO to form adducts which are stable over a period of weeks. The same species can be formed in higher yields by careful reduction of the ferrous nitrosyl adducts of the proteins. Like the analogous O(2)-Fe(II) adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in (1)H NMR at ca. 15 ppm that splits into doublets for H(15)NO adducts. The (1)H and (15)N NMR resonances, obtained by HSQC experiments, are shown to differentiate subunits and isoforms of proteins within mixtures. An apparent difference in the reduction rates of the NO adducts of the two subunits of human hemoglobin allows assignment of two distinct nitrosyl hydride peaks by a combination of UV-vis, NMR, and EPR analysis. The two peaks of the HNO-hHb adduct have a persistent 3:1 ratio during trapping reactions, demonstrating a kinetic difference between HNO binding at the two subunits. These results show NMR characterization of ferrous HNO adducts as a unique tool sensitive to structural changes within the oxygen-binding cavity, which may be of use in defining modes of oxygen binding in other heme proteins and enzymes.

Chemical trapping and characterization of small oxoacids of sulfur (SOS) generated in aqueous oxidations of H2S.

Small oxoacids of sulfur (SOS) are elusive molecules like sulfenic acid, HSOH, and sulfinic acid, HS(O)OH, generated during the oxidation of hydrogen sulfide, H2S, in aqueous solution. Unlike their alkyl homologs, there is a little data on their generation and speciation during H2S oxidation. These SOS may exhibit both nucleophilic and electrophilic reactivity, which we attribute to interconversion between S(II) and S(IV) tautomers. We find that SOS may be trapped in situ by derivatization with nucleophilic and electrophilic trapping agents and then characterized by high resolution LC MS. In this report, we compare SOS formation from H2S oxidation by a variety of biologically relevant oxidants. These SOS appear relatively long lived in aqueous solution, and thus may be involved in the observed physiological effects of H2S.

Trapping Reactions of the Sulfenyl and Sulfinyl Tautomers of Sulfenic Acids.

Sulfenic acids react as both nucleophiles and electrophiles, which may be attributable to interconversion between sulfenyl and sulfinyl tautomers. We demonstrate one-pot trapping of both tautomeric forms of glutathione sulfenic acid by LCMS. The sulfinyl tautomers are characterized by reaction with nucleophilic reagents such as dimedone and cyanide, giving unique products that are analogous to corresponding adducts of aldehydes. Likewise, we show that aldehyde reactive reagents such as silyl enol ethers also react with glutathione sulfenic acid to give products characteristic of both sulfenyl and sulfinyl tautomers.

A singular value decomposition approach for kinetic analysis of reactions of HNO with myoglobin.

The reactions of several horse heart myoglobin species with nitrosyl hydride, HNO, derived from Angeli's salt (AS) and Piloty's acid (PA) have been followed by UV-visible, (1)H NMR and EPR spectroscopies. Spectral analysis of myoglobin-derived speciation during the reactions was obtained by using singular value decomposition methods combined with a global analysis to obtain the rate constants of complex sequential reactions. The analysis also provided spectra for the derived absorbers, which allowed self-consistent calibration to the spectra of known myoglobin species. Using this method, the determined rate for trapping of HNO by metmyoglobin, which produces NO-myoglobin, is found to be 2.7 × 10(5)M(-1)s(-1) at pH7.0 and 1.1 × 10(5)M(-1)s(-1) at pH9.4. The reaction of deoxymyoglobin with HNO generates the adduct HNO-myoglobin directly, but is followed by a secondary reaction of that product with HNO yielding NO-myoglobin; the determined bimolecular rate constants for these reactions are 3.7 × 10(5)M(-1)s(-1) and 1.67 × 10(4)M(-1)s(-1) respectively, and are independent of pH. The derived spectrum for HNO-myoglobin is characterized by a Soret absorbance maximum at 423 nm with an extinction coefficient of 1.66 × 10(5)M(-1)cm(-1). The rate constant for unimolecular loss of HNO from HNO-myoglobin was determined by competitive trapping with CO at 8.9 × 10(-5)s(-1), which gives the thermodynamic binding affinity of HNO to deoxymyoglobin as 4.2 × 10(9)M(-1). These results suggest that the formation of HNO-ferrous heme protein adducts represents an important consideration in the biological action of HNO-releasing drugs.