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.

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.

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 [...].

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.

Characterization of the Initial Intermediate Formed during Photoinduced Oxygenation of the Ruthenium(II) Bis(bipyridyl)flavonolate Complex.

A ruthenium(II) flavonolate complex, [Ru(II)(bpy)2fla][BF4], was synthesized to model the reactivity of the flavonol dioxygenases. The treatment of dry CH3CN solutions of [Ru(II)(bpy)2fla][BF4] with dioxygen under light leads to the oxidative O-heterocyclic ring opening of the coordinated substrate flavonolate, resulting in the formation of [Ru(II)(bpy)2(carboxylate)][BF4] (carboxylate = O-benzoylsalicylate or benzoate) species, as determined by electrospray ionization mass spectrometry. Moderation of the excitation and temperature allowed isolation and characterization of an intermediate, [Ru(II)(bpy)2bpg][BF4] (bpg = 2-benzoyloxyphenylglyoxylate), generated by the 1,2-addition of dioxygen to the central flavonolate ring.

Amyloids, melanins and oxidative stress in melanomagenesis.

Melanoma has traditionally been viewed as an ultraviolet (UV) radiation-induced malignancy. While UV is a common inducing factor, other endogenous stresses such as metal ion accumulation or the melanin pigment itself may provide alternative pathways to melanoma progression. Eumelanosomes within melanoma often exhibit disrupted membranes and fragmented pigment which may be due to alterations in their amyloid-based striated matrix. The melanosomal amyloid can itself be toxic, especially in combination with reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by endogenous NADPH oxidase (NOX) and nitric oxide synthase (NOS) enzymes, a toxic mix that may initiate melanomagenesis. Further understanding of the loss of the melanosomal organization, the behaviour of the exposed melanin and the induction of ROS/RNS in melanomas may provide critical insights into this deadly disease.

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.

Kinetic characterization of a slow-binding inhibitor of Bla2: thiomaltol.

The increasing prevalence of drug resistant bacteria is a pandemic problem. Metallo-ß-lactamases (MBLs) are one of the main causes of drug resistance due to hydrolysis of ß-lactam antibiotics. Thus, the development of effective inhibitors of MBLs remains urgent. The compound thiomaltol was used as a lead compound to investigate its ability to inhibit metallo-ß-lactamase from Bacillus anthracis (Bla2), which causes anthrax. Kinetic evaluation with nitrocefin as a substrate indicates that thiomaltol inhibits Bla2 in a time-dependent manner with an IC(50) value of 290 µM after 20 min preincubation. Progress curve analysis and reversibility tests suggest that thiomaltol is a reversible, slow-binding inhibitor with a K(i) of 85 ± 30 µM. Furthermore, studies on the modality of inhibition and in silico analysis indicate thiomaltol to be a competitive inhibitor. The results demonstrate that thiomaltol is a promising lead compound for slow binding inhibitor design of Bla2.

The case for an oxidopyrylium intermediate in the mechanism of quercetin dioxygenases.

The quercetin dioxygenases (QDOs) are unusual metalloenzymes in that they display ring-opening dioxygenase activity with several different first-row transition metal ions which do not undergo redox changes during turnover. The QDOs are also unique in that the substrate binds as an η1-flavonolate rather than the η2 -bidentate mode seen in all reported model complexes. The flavonol substrates were early examples of excited state intramolecular proton transfer (ESIPT) phenomena, in which photoexcitation causes an H-atom exchange between the adjacent hydroxyl and ketone, generating an oxidopyrylium emissive state. These oxidopyryliums undergo ring-opening dioxygenations analogous to the enzymatic reactions. Our hypothesis is that lability of the divalent metal ion may allow access to a reactive oxidopyrylium intermediate via coordination switching from the oxy to ketone position, which allows reaction with O2. In this report, we use a straight-forward methylation strategy to generate a panel of flavonol and thioflavonol derivatives modeling several η1- and η2-coordination modes. Methylation of 3-hydroxythioflavone generates an air stable η1 hydroxopyrylium salt, which undergoes rapid ring-opening dioxygenation by deprotonation or photoexcitation. By comparison, the η1-methoxyflavonol does not react with O2 under any condition. We find that any of the studied flavonol derivatives, η1 or η2, which demonstrates ESIPT-like oxidopyrylium emissions undergo QDO-like ring-opening reactions with dioxygen. The implications of these results concerning the mechanism of QDOs and related dioxygenases is discussed.

Probing the motional behavior of eumelanin and pheomelanin with solid-state NMR spectroscopy: new insights into the pigment properties.

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV-radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu- and pheomelanin extracted from black and red human hair are investigated by means of solid-state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the (1) H NMR wide-line spectra extracted from 2D (1) H-(13) C LG-WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu- and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D (1) H-(13) C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.

Direct oxygen imaging in titania nanocrystals.

Recently, rutile nanotwins were synthesized using high temperature organic solvent methods, yielding two kinds of common high-quality rutile twinned nanocrystals, (101) and (301) twins, accompanied by minor rutile nanorods (Lu et al 2012 CrystEngComm 14 3120-4). In this report, the atomic structures of the rutile and anatase nanocrystals are directly resolved with no need for calculation or image simulation using atomic resolution STEM techniques. The locations of the oxygen rows in the rutile twins' boundaries are directly determined from both HAADF images and ABF images. To the best of our knowledge, this is the first time oxygen columns have been distinguished in rutile twin boundaries using HAADF and BF imaging.

The ionophore thiomaltol induces rapid lysosomal accumulation of copper and apoptosis in melanoma.

In this report, we investigate the toxicity of the ionophore thiomaltol (Htma) and Cu salts to melanoma. Divalent metal complexes of thiomaltol display toxicity against A375 melanoma cell culture resulting in a distinct apoptotic response at submicromolar concentrations, with toxicity of Cu(tma)2 > Zn(tma)2 >> Ni(tma)2. In metal-chelated media, Htma treatment shows little toxicity, but the combination with supplemental CuCl2, termed Cu/Htma treatment, results in toxicity that increases with suprastoichiometric concentrations of CuCl2 and correlates with the accumulation of intracellular copper. Electron microscopy and confocal laser scanning microscopy of Cu/Htma treated cells shows a rapid accumulation of copper within lysosomes over the course of hours, concurrent with the onset of apoptosis. A buildup of ubiquitinated proteins due to proteasome inhibition is seen on the same timescale and correlates with increases of copper without additional Htma.

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.

Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii.

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe2S2] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP+ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (Em = -321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.

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.

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma.

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.

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.

Synthesis of first row transition metal selenomaltol complexes.

We report an efficient, one-step synthesis of the chelator 3-hydroxy-2-methyl-4-selenopyrone (selenomaltol). Complexes of selenomaltol with Fe(iii), Ni(ii), Cu(ii) and Zn(ii) have been prepared and studied by NMR, X-ray crystallography, cyclic voltammetry, EPR and electronic absorption. The Ni(ii) and Cu(ii) complexes show chemically reversible oxidations which are suggested to be ligand-based. Nuclear independent chemical shifts (NICS) analysis is used to compare aromaticity of the heterocyclic rings of selenomaltol and its chelates. The compounds described here should significantly expand the scope and utility of unusual O,Se-donor chelates.