Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: a review.

Hydrogen sulfide is rapidly emerging as an important vasoactive mediator formed in health and disease. Its biological action is centered on its reactivity with heme-proteins and its ability to activate K(ATP) channels. Hydrogen sulfide is a signalling molecule of the inflammatory and nervous systems, and in particular the cardiovascular system where it regulates vascular tone, cardiac work, and exerts cardioprotection. This has led to an explosion of papers in which the role of hydrogen sulfide generated in vitro has been used to stimulate biological responses, and where a variety of methods have been used to measure the concentration of this compound in biological fluids. Understanding the chemistry and the inherent problems in the analytical techniques used to measure hydrogen sulfide concentrations is critical to our expanding knowledge on the biology of hydrogen sulfide. In this brief review we will cover the chemistry of hydrogen sulfide, including sources of hydrogen sulfide, its speciation at physiological pH, the susceptibility of sulfide to aerobic oxidation, and the methods used to measure hydrogen sulfide concentrations in solution, including biological fluids. We also give a brief overview of knockout animals and inhibition of the enzymes involved in the formation of hydrogen sulfide in vivo.

Chemistry of nitric oxide and related species.

Nitric oxide (NO) has essential roles in a remarkable number of diverse biological processes. The reactivity of NO depends upon its physical properties, such as its small size, high diffusion rate, and lipophilicity (resulting in its accumulation in hydrophobic regions), and also on its facile but selective chemical reactivity toward a variety of cellular targets. NO also undergoes reactions with oxygen, superoxide ions, and reducing agents to give products that themselves show distinctive reactivity toward particular targets, sometimes with the manifestation of toxic effects, such as nitrosative stress. These include nitroxyl (HNO), the oxides NO2/N2O4, and N2O3, peroxynitrite, and S-nitrosothiols (RSNO). HNO is attracting considerable attention due to its pharmacological properties, which appear to be distinct from those of NO, and that may be significant in the treatment of heart failure.

The preparation and purification of NO gas and the use of NO releasers: the application of NO donors and other agents of nitrosative stress in biological systems.

Cylinders and lecture bottles are often the source of nitric oxide (NO) in studies of the biological chemistry of this remarkable molecule. The NO from both sources will probably contain NO2 (and N2O) formed by disproportionation of NO. The NO2 must be removed by passing the NO through a thoroughly deoxygenated sequence of traps containing sodium hydroxide solution and then water. The presence of NO2 in aqueous solutions of NO may be determined readily using 2,2' azino(3-ethylbenzothiazoline-6-sulfonic acid). NO2 oxidizes this compound to a long-lived radical anion, the concentration of which may be determined spectrophotometrically. The formation of NO by the decomposition of nitrous acid (via its disproportionation to nitrate and NO) and by the use of commercially available NO-releasing compounds with defined half-lives are also discussed. Other reactions that lead indirectly to the formation of NO are noted. In all cases, care must be taken to exclude oxygen to minimize as much as possible the formation of NO2 (and, consequently, the nitrosating agent N2O3). The uses of these methods for generating NO and the reactivity of related compounds are illustrated with examples of studies of nitrosative stress.

Inhibitory effects of sulfamic acid on three thiosulfate-oxidizing chemolithotrophs.

Sulfamate is an analogue of thiosulfate, and the sodium and potassium salts of sulfamic acid inhibited the chemolithoautotrophic growth on thiosulfate of Acidithiobacillus ferrooxidans and Halothiobacillus neapolitanus. The chemo-organotrophic growth of Paracoccus versutus on sucrose was similarly inhibited by sulfamate. Thiosulfate oxidation by suspensions of H. neapolitanus was, however, unaffected by sulfamate, showing that sulfamate did not directly affect thiosulfate uptake, activation or oxidation. Inhibition of P. versutus was not relieved by cysteine and methionine, indicating that sulfate uptake and sulfur amino acid biosynthesis were not directly affected by sulfamate. Sulfamate was not degraded by any of the bacteria, and so could not serve as an alternative to thiosulfate as an energy-yielding substrate. Sulfamate is also an analogue of ammonia and might act like hydrazine by inhibiting ammonium uptake or an essential enzyme activity.

Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis.

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 microm GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.

Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions.

The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by live bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by live bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia.

Interaction of bilirubin and biliverdin with reactive nitrogen species.

Bilirubin (BR) and biliverdin (BV), two metabolites produced during haem degradation by haem oxygenase, possess strong antioxidant activities toward peroxyl radical, hydroxyl radical and hydrogen peroxide. Considering the importance attributed to nitric oxide (NO) and its congeners in the control of physiological and pathophysiological processes, we examined the interaction of BR and BV with NO and NO-related species in vitro. Exposure of BR and BV to agents that release NO or nitroxyl resulted in a concentration- and time-dependent loss of BR and BV, as assessed by high performance liquid chromatography. Peroxynitrite, a strong oxidant derived from the reaction of NO with superoxide anion, also showed high reactivity toward BR and BV. The extent of BR and BV consumption largely depended on the NO species being analysed and on the half-lives of the pharmacological compounds considered. Of major importance, BR and BV decomposition occurred also in the presence of pure NO under anaerobic conditions, confirming the ability of bile pigments to scavenge the gaseous free radical. Increasing concentrations of thiols prevented BR consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergise the cellular defence against NO-related species. In view of the high inducibility of haem oxygenase-1 by NO-releasing agents in different cell types, the present findings highlight novel anti-nitrosative characteristics of BR and BV suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production.

NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp.

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O(2)-responsive regulator FNR, we investigated whether FNR also senses NO. The [4Fe-4S](2+) cluster of FNR is oxygen labile and controls protein dimerization and site-specific DNA binding. NO reacts anaerobically with the Fe-S cluster of purified FNR, generating spectral changes consistent with formation of a dinitrosyl-iron-cysteine complex. NO-inactivated FNR can be reconstituted, suggesting physiological relevance. FNR binds at an FNR box within the hmp promoter (P(hmp)). FNR samples inactivated by either O(2) or NO bind specifically to P(hmp), but with lower affinity. Dose-dependent up-regulation of P(hmp) in vivo by NO concentrations of pathophysiological relevance is abolished by fnr mutation, and NO also modulates expression from model FNR-regulated promoters. Thus, FNR can respond to not only O(2), but also NO, with major implications for global gene regulation in bacteria. We propose an NO-mediated mechanism of hmp regulation by which E.coli responds to NO challenge.

Nitric oxide releases intracellular zinc from prokaryotic metallothionein in Escherichia coli.

Nitric oxide (NO) has a broad spectrum of signalling and regulatory functions and multiple molecular targets. Recently, the intrabacterial toxicity of NO and mechanisms for NO resistance have been intensively investigated. Here we report for the first time that NO elicits release of zinc from a bacterial protein. Using the zinc-responsive expression of zntA (encoding a Zn-exporting P-type ATPase) fused to lacZ, i.e. Phi(zntA-lacZ), to monitor intracellular zinc, and SmtA (the Synechococcus metallothionein) as zinc store, we have shown that the NO donors NOC-5 and NOC-7 elicit zinc ejection. No increase in Phi(zntA-lacZ) activity was observed in a zntR mutant, indicating the specificity of the zntA promoter response to zinc ions.

Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH.

Nitrogen dioxide (NO(2)(*)) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO(2)(*) were generated in < 1 micros using pulse radiolysis, and the kinetics of scavenging NO(2)(*) by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO(2)(*) with the thiols (the thiyl radical) was monitored via oxidation of 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO(2)(*) with glutathione, cysteine, and urate were estimated as approximately 2 x 10(7), 5 x 10(7), and 2 x 10(7) M(-1) s(-1), respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO(2)(*) at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of approximately 3 x 10(7) M(-1) s(-1). The implications of these values are: (i) the lifetime of NO(2)(*) in cytosol is < 10 micros; (ii) thiols are the dominant 'sink' for NO(2)(*) in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO(2)(*) is approximately 0.2 microm in the cytoplasm and < 0.8 microm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO(2)(*); and (v) reactions between NO(2)(*) and thiols/urate severely limit the likelihood of reaction of NO(2)(*) with NO* to form N(2)O(3) in the cytoplasm.

The reaction of superoxide radicals with S-nitrosoglutathione and the products of its reductive heterolysis.

Generation of superoxide radicals (0.01-0.1 microm s(-1)) by radiolysis of aqueous solutions containing S-nitrosoglutathione (45-160 microm, pH 3.8-7.3) resulted in loss of this solute at rates varying with solute concentration, radical generation rate, and pH. The results were quantitatively consistent with the loss being attributed to competition between reaction of superoxide with S-nitrosoglutathione (rate constant 300 +/- 100 m(-1) s(-1)) and the pH-dependent disproportionation of superoxide/hydroperoxyl. This rate constant is much lower than previous estimates and seven orders of magnitude lower than the rate constants between superoxide and superoxide dismutase or superoxide and nitric oxide. This indicates that interaction between superoxide and S-nitrosoglutathione is unlikely to be biologically important, contrary to previous suggestions that reaction could serve to prevent the rapid reaction between superoxide and nitric oxide. Reductive homolysis of S-nitrosoglutathione by the carbon dioxide radical anion, a model for biological reductants such as disulfide radical anions, occurred with a rate constant of 7.4 x 10(8) m(-1) s(-1) and produced nitric oxide stoichiometrically. Thiyl radicals were not produced, indicating the alternative homolysis route to generate nitroxyl did not occur.

Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen.

Escherichia coli flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of O2 reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0.5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(III)-hydroxamate K from Rhizobium leguminosarum bv. viciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH-cytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of O2 at the haem. The Vmax of cytochrome c reduction fell from 7.1 s-1 in the presence of 0.5 mM CO to 5.0 s-1 in the presence of 100 microM O2, with no significant change in K(m) for cytochrome c (6.8 to 7.3 microM, respectively). O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 microM O2. Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globin-like haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of O2 since, in the absence of O2, electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in vivo is not known and alternative models for O2 sensing are also considered.