Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Assessment of Landfill Leachate Volume and Concentrations of Cyanide and Fluoride during Phytoremediation

The potential for plants to minimize leachate volume and reduce cyanide and fluoride concentrations in groundwater was evaluated. High fluoride and soluble salts in the leachate induced chlorosis or necrosis in the leaf margins on green ash (Fraxinus pennsylvanica), yellow poplar (Liriodendron tulipifera), and bald cypress (Taxodium distichum). Hybrid willow (Salix Willow hybrid), sycamore (Platanus sp.), and black willow (Salix nigra) had high rates of transpiration and root growth during the study period. Cyanide in the leachate was removed by plant metabolic processes whereas fluoride accumulated in the leaves. Cyanide and fluoride in landfill leachate can be decreased through phytoremediation.     

Protein synthesis in isolated castor bean mitochondria is stimulated by cyanide

Cyanide added to isolated castor bean (Ricinus communis L.) mitochondria supplemented with ATP and succinate (or NADH) significantly enhanced the rate and extent of organellar protein synthesis. Cyanide stimulated mitochondrial protein synthesis in a dose-dependent manner with an optimum stimulation of over twofold at 1 millimolar cyanide. At this concentration of cyanide, the mitochondrial respiratory activity, in the presence of succinate (or NADH) and ADP was inhibited by 90%. The stimulatory effect of cyanide on mitochondrial translation was reflected in the increased synthesis of all the proteins synthesized within the organelle. Preliminary evidence indicates a role for the alternative, salicylhydroxamic acid-sensitive, oxidase in the cyanide stimulation of protein synthesis.     

THE REACTIONS OF CYANIDE WITH GLOBIN HEMOCHROMOGEN

Cyanide can react with globin hemochromogen in two different ways. In the first reaction cyanide combines with globin hemochromogen without displacing or competing with globin. In the second reaction cyanide displaces globin.     

Exploring anaerobic environments for cyanide and cyano-derivatives microbial degradation

Applied Microbiology and Biotechnology - Cyanide is one of the most toxic chemicals for living organisms described so far. Its toxicity is mainly based on the high affinity that cyanide presents...     

Cyanide insensitive palmitate oxidation in skeletal muscle

Cyanide insensitive palmitate oxidation is demonstrated in rat gastroc and human biceps using radioisotope labeled palmitate. Similar activity is confirmed in rat liver. The implication for studies of fatty acid oxidation in skeletal muscle as well as for human muscle illnesses is discussed.     

Quantitative similarities in the several actions of cyanide on prostaglandin H synthase

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a KI value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflected an increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a KI of 4 mM. The higher KI value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

The VI international conference on prostaglandins and related compounds

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a K_I value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflect and increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a K_I of 4 mM. The higher K_I value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 involves a putative siderophore

Cyanide utilization in P. fluorescens NCIMB 11764 requires the induction of cyanide oxygenase. The enzyme is induced during growth on cyanide as the sole nitrogen source or when cyanide is added to stationary-phase cells grown on limiting ammonia, however, enzyme induction and cyanide degradation were found to occur non-concomitantly. Cyanide removal by chloramphenicol-treated cells and a mutant strain (JL102) defective in cyanide oxygenase were also observed. We now report a non-enzymatic mechanism for cyanide biotransformation by an iron-chelating species identified as a putative siderophore. Partially purified siderophore preparations removed cyanide at initial rates as high as 7.6 mmol min⁻¹ mg⁻¹. The reaction product was further shown to be enzymatically oxidized to NH₃ (and CO₂) and also supported growth. The results indicate that both cyanide oxygenase and a putative siderophore component are involved in cyanide utilization.     

Hypoxic Excitability Changes and Sodium Currents in Hippocampus CA1 Neurons

1. The objective of the present study was to distinguish if inhibition of neuronal activity by hypoxia is related to a block of voltage-gated Na+ channels. 2. The effect of chemical hypoxia induced by cyanide (0.5 mM, 10 min perfusion) was studied with patch-clamp technique in visualized intact CA1 pyramidal neurons in rat brain slices. Action potentials were elicited in whole cell current-clamp recordings and the threshold was estimated by current pulses of 50-ms duration and incremental amplitudes (n = 31). The effect of cyanide on the Na+ current and conductance was studied in voltage clamp recordings from cell-attached patches (n = 13). 3. Cyanide perfusion during 10 min increased the threshold for excitation by 73 +/- 79 pA (p = 0.001), which differed from the effect in control cells (11 +/- 41 pA, ns). The change in current threshold was correlated to a change in membrane potential (r = -0.88, p < 0.0001). Cyanide had no significant effect on the peak amplitude, duration, or rate of rise of the action potential. 4. Cyanide perfusion did not change the Na+ current size, but caused a small decrease in ENa (-17 +/- 22 mV, ns) and a slight increase in Na+ conductance (+14 +/- 26%, ns), which differed (p = 0.045) from controls (-19 +/- 23 %, ns). 5. In conclusion, chemical hypoxia does not cause a decrease in Na+ conductance. The decreased excitability during hypoxia can be explained by an increase in the current threshold, which is correlated with the effect on the membrane potential.     

Cyanide binding to cd(1) nitrite reductase from Pseudomonas aeruginosa: role of the active-site His369 in ligand stabilization

Cyanide binding to fully reduced Pseudomonas aeruginosa cd(1) nitrite reductase (Pa cd(1) NiR) has been investigated for the wild-type enzyme and a site-directed mutant in which the active-site His369 was replaced by Ala. This mutation reduces the affinity toward cyanide (by approximately 13-fold) and especially decreases the rate of binding of cyanide to the reduced d(1) heme (by approximately 100-fold). The crystal structure of wild-type reduced Pa cd(1) NiR saturated with cyanide was determined to a resolution of 2.7 A. Cyanide binds to the iron of the d(1) heme, with an Fe-C-N angle of 168 degrees for both subunits of the dimer and only His369 is within hydrogen bonding distance of the nitrogen atom of the ligand. These results suggest that in Pa cd(1) NiR the invariant distal residue His369 plays a dominant role in controlling the binding of anionic ligands and allow the discussion of the mechanism of cyanide binding to the wild-type enzyme.     

Spectroscopic and ligand-binding properties of an oxygen-binding heme protein from Chromatium vinosum

Magnetic circular dichroism spectra were obtained for the oxidized and reduced forms of cyanide, azide and carbon monoxide complexes of an O2-binding hemeprotein isolated from the photosynthetic purple sulfur bacterium, Chronatium vinosum. Cyanide binding to the protein, which results in formation of a low-spin complex, was highly pH dependent with little complex formation observed at pH values near or below 7.     

Cyanide degradation by immobilised fungi

Summary Cyanide hydratase, which converts cyanide to formamide, was induced in mycelia of Stemphylium loti by growth in the presence of low concentrations of cyanide. Mycelia were immobilised by several methods. The most useful system was found to be treatment with flocculating agents. This technique is applicable to a wide range of easily isolated fungi that contain cyanide hydratase.     

Nickel-specific, slow-binding inhibition of carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.     

Method for Rapid Detection of Cyanogenic Bacteria

An agar plate method is described in which the production of hydrogen cyanide by as many as 50 microbial isolates per plate may be detected. Cyanide produced by the organisms reacts with copper(II) ethylacetoacetate and 4,4′-methylenebis-(N,N-dimethylaniline) in a paper disk suspended above the microbial colonies. Cell growth occurs in depressions in the agar surface, which allows separation of colonies and enhances sensitivity of hydrogen cyanide detection.     

Evidence for cyanide and mercury inactivation of endogenous plastocyanin

Cyanide and mercury treatment of chloroplast membranes inactivates plastocyanin as shown by the inability of the extracted plastocyanin to restore electron transport in a bioassay on chloroplasts depleted of their endogenous plastocyanin by digitonin treatment. The extraction procedure did remore the enzyme from cyanide and mercury treated chloroplasts as shown by sodium dodecyl sulfate polyacrylamide electrophoresis of the extracts. This procedure normally shows a plastocyanin band at 11,000 dalton molecular weight and the band was present in extracts from control and cyanide or mercury treated membranes.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Cyanide interaction with redox modulatory sites enhances NMDA receptor responses.

Activation of NMDA receptors plays an important role in cyanide neurotoxicity. Cyanide indirectly activates the receptor by inducing neuronal release of glutamate and also enhances receptor-mediated responses by a direct interaction with the receptor complex. This study investigated the mechanism in cerebellar granule cells by which cyanide enhances NMDA-induced Ca2+ influx. Cyanide (50 microM) increased the influx of Ca2+ over the NMDA concentration range of 0.5-500 microM. Experiments showed that cyanide does not interact with the receptor's glycine or PKC mediated phosphorylation regulatory sites. N-ethylmaleimide, a thiol alkylating agent which inactivates the redox regulatory sites of the receptor, blocked the enhancing effect of cyanide. Pretreatment of cells with 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), a compound that oxidizes the receptor redox sites, had no effect on the response to cyanide. On the other hand, the nonpermeant reducing agents, dithiothreitol or cysteine, further increased the cyanide effect. These observations can be explained by cyanide interacting with redox sensitive disulfide groups that are not accessible to the non-permeant reducing agents. It is proposed that cyanide interacts with a redox site(s) located either on the intracellular receptor domain or in the transmembrane hydrophobic domain. Furthermore the enhancement by cyanide of the excitotoxic actions of NMDA involves receptor sites that are sensitive to oxidation/reduction and this interaction contributes to the neurotoxic action of cyanide.