Heme-Thiolate perturbation in cystathionine β-Synthase by mercury compounds

Cystathionine β-synthase (CBS) is an enzyme involved in sulfur metabolism that catalyzes the pyridoxal phosphate-dependent condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. CBS possesses a b-type heme coordinated by histidine and cysteine. Fe(III)-CBS is inert toward exogenous ligands, while Fe(II)-CBS is reactive. Both Fe(III)- and Fe(II)-CBS are sensitive to mercury compounds. In this study, we describe the kinetics of the reactions with mercuric chloride (HgCl2) and p-chloromercuribenzoic acid. These reactions were multiphasic and resulted in five-coordinate CBS lacking thiolate ligation, with six-coordinate species as intermediates. Computational QM/MM studies supported the feasibility of formation of species in which the thiolate is proximal to both the iron ion and the mercury compound. The reactions of Fe(II)-CBS were faster than those of Fe(III)-CBS. The observed rate constants of the first phase increased hyperbolically with concentration of the mercury compounds, with limiting values of 0.3–0.4 s–1 for Fe(III)-CBS and 40 ± 4 s–1 for Fe(II)-CBS. The data were interpreted in terms of alternative models of conformational selection or induced fit. Exposure of Fe(III)-CBS to HgCl2 led to heme release and activity loss. Our study reveals the complexity of the interactions between mercury compounds and CBS

Heme-Thiolate Perturbation in Cystathionine β-Synthase by Mercury Compounds

Cystathionine β-synthase (CBS) is an enzyme involved in sulfur metabolism that catalyzes the pyridoxal phosphate-dependent condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. CBS possesses a b-type heme coordinated by histidine and cysteine. Fe(III)-CBS is inert toward exogenous ligands, while Fe(II)-CBS is reactive. Both Fe(III)-and Fe(II)-CBS are sensitive to mercury compounds. In this study, we describe the kinetics of the reactions with mercuric chloride (HgCl2) and p-chloromercuribenzoic acid. These reactions were multiphasic and resulted in five-coordinate CBS lacking thiolate ligation, with six-coordinate species as intermediates. Computational QM/MM studies supported the feasibility of formation of species in which the thiolate is proximal to both the iron ion and the mercury compound. The reactions of Fe(II)-CBS were faster than those of Fe(III)-CBS. The observed rate constants of the first phase increased hyperbolically with concentration of the mercury compounds, with limiting values of 0.3-0.4 s-1 for Fe(III)-CBS and 40 ± 4 s-1 for Fe(II)-CBS. The data were interpreted in terms of alternative models of conformational selection or induced fit. Exposure of Fe(III)-CBS to HgCl2 led to heme release and activity loss. Our study reveals the complexity of the interactions between mercury compounds and CBS.Fil: Benchoam, Dayana. Universidad de la Republica; UruguayFil: Cuevasanta, Ernesto. Universidad de la Republica; UruguayFil: Julió Plana, Laia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Capece, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Banerjee, Ruma. University of Michigan; Estados UnidosFil: Alvarez, Beatriz. Universidad de la República; Urugua

Kinetics of nitrite reduction and peroxynitrite formation by ferrous heme in human cystathionine β-synthase

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO·), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2 to Fe(III)-CBS, forming superoxide radical anion (O2 -·). In this study, we describe the kinetics of nitrite (NO2·-) reduction by Fe(II)-CBS to form Fe(II)NO·-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO·-CBS by O2 showed complex kinetic behavior and led to peroxynitrite (ONOO-) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO· and peroxynitrite

Possible molecular basis of the biochemical effects of cysteine-derived persulfides

Persulfides (RSSH/RSS−) are species closely related to thiols (RSH/RS−) and hydrogen sulfide (H2S/HS−), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed

Fluorescent detection of hydrogen sulfide (H2S) through the formation of pyrene excimers enhances H2S quantification in biochemical systems

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.MEC: I/FVF2017/069ANII: FCE_1_2017_1_136043CSIC: I+D 2017; I+D 202

Acidity and nucleophilic reactivity of glutathione persulfide

Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/ GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the a effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.Fil: Benchoam, Dayana. Universidad de la República; UruguayFil: Semelak, Jonathan Alexis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Mastrogiovanni, Mauricio. Universidad de la Republica; UruguayFil: Grassano, Juan S.. Universidad de Buenos Aires; ArgentinaFil: Ferrer-Sueta, Gerardo. Universidad de la Republica; UruguayFil: Zeida Camacho, Ari Fernando. Universidad de la Republica; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Möller, Matías N.. Universidad de la Republica; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Alvarez, Beatriz. Universidad de la Republica; Urugua

Solubility and Permeation of Hydrogen Sulfide in Lipid Membranes

Hydrogen sulfide (H2S) is mainly known for its toxicity but has recently been shown to be produced endogenously in mammalian tissues and to be associated with physiological regulatory functions. To better understand the role of biomembranes in modulating its biological distribution and effects; we measured the partition coefficient of H2S in models of biological membranes. The partition coefficients were found to be 2.1±0.2, 1.9±0.5 and 2.0±0.6 in n-octanol, hexane and dilauroylphosphatidylcholine liposome membranes relative to water, respectively (25°C). This two-fold higher concentration of H2S in the membrane translates into a rapid membrane permeability, Pm = 3 cm s−1. We used a mathematical model in three dimensions to gain insight into the diffusion of total sulfide in tissues. This model shows that the sphere of action of sulfide produced by a single cell expands to involve more than 200 neighboring cells, and that the resistance imposed by lipid membranes has a significant effect on the diffusional spread of sulfide at pH 7.4, increasing local concentrations. These results support the role of hydrogen sulfide as a paracrine signaling molecule and reveal advantageous pharmacokinetic properties for its therapeutic applications

Hydrogen Sulfide and Persulfides Oxidation by Biologically Relevant Oxidizing Species

Hydrogen sulfide (H2S/HS⁻) can be formed in mammalian tissues and exert physiological effects. It can react with metal centers and oxidized thiol products such as disulfides (RSSR) and sulfenic acids (RSOH). Reactions with oxidized thiol products form persulfides (RSSH/RSS⁻). Persulfides have been proposed to transduce the signaling effects of H2S through the modification of critical cysteines. They are more nucleophilic and acidic than thiols and, contrary to thiols, also possess electrophilic character. In this review, we summarize the biochemistry of hydrogen sulfide and persulfides, focusing on redox aspects. We describe biologically relevant one- and two-electron oxidants and their reactions with H2S and persulfides, as well as the fates of the oxidation products. The biological implications are discussed

Heme-Thiolate Perturbation in Cystathionine β-Synthase by Mercury Compounds

Cystathionine β-synthase (CBS) is an enzyme involved in sulfur metabolism that catalyzes the pyridoxal phosphate-dependent condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. CBS possesses a b-type heme coordinated by histidine and cysteine. Fe(III)-CBS is inert toward exogenous ligands, while Fe(II)-CBS is reactive. Both Fe(III)- and Fe(II)-CBS are sensitive to mercury compounds. In this study, we describe the kinetics of the reactions with mercuric chloride (HgCl2) and p-chloromercuribenzoic acid. These reactions were multiphasic and resulted in five-coordinate CBS lacking thiolate ligation, with six-coordinate species as intermediates. Computational QM/MM studies supported the feasibility of formation of species in which the thiolate is proximal to both the iron ion and the mercury compound. The reactions of Fe(II)-CBS were faster than those of Fe(III)-CBS. The observed rate constants of the first phase increased hyperbolically with concentration of the mercury compounds, with limiting values of 0.3–0.4 s–1 for Fe(III)-CBS and 40 ± 4 s–1 for Fe(II)-CBS. The data were interpreted in terms of alternative models of conformational selection or induced fit. Exposure of Fe(III)-CBS to HgCl2 led to heme release and activity loss. Our study reveals the complexity of the interactions between mercury compounds and CBS