Quantitation and Characterization of CytochromecOxidase in Complex Systems

Quantitation of cytochromecoxidase in complex systems such as tissue homogenates is often hampered by the presence of other hemoproteins. Cyanide can bind to reduced cytochromecoxidase from diverse sources with a dissociation constant in the range of 0.1–0.5 mM and induces a characteristic optical change. This contrasts with the very weak binding of cyanide to reduced forms of many other hemoproteins, including hemoglobin and myoglobin. Hence, difference spectra of cyanide binding to reduced samples can provide an improved method to resolve and quantitate cytochromecoxidase. In addition, the cyanide compound of cytochromecoxidase is photolabile. This property can be exploited to further enhance the sensitivity of detection and analysis of cytochromecoxidase.     

The Cold Origin of Life: A. Implications Based On The Hydrolytic Stabilities Of Hydrogen Cyanide And Formamide

It has been suggested that hydrogen cyanide(HCN) would not have been present in sufficient concentrationto polymerize in the primitive ocean to produce nucleic acidbases and amino acids. We have measured the hydrolysis ratesof HCN and formamide over the range of 30–150 °C and pH 0–14,and estimated the steady state concentrations in theprimitive ocean. At 100 °C and pH 8, the steady stateconcentration of HCN and formamide were calculated to be7 × 10^-13 M and 1 × 10^-15 M, respectively. Thus, itseems unlikely that HCN could have polymerized in a warmprimitive ocean. It is suggested that eutectic freezing mighthave been required to have concentrated HCN sufficiantly forit to polymerize. If the HCN polymerization was important forthe origin of life, some regions of the primitive earth mighthave been frozen.     

Was ferrocyanide a prebiotic reagent?

Hydrogen cyanide is the starting material for a diverse array of prebiotic syntheses, including those of amino acids and purines. Hydrogen cyanide also reacts with ferrous ions to give ferrocyanide, and so it is possible that ferrocyanide was common in the early ocean. This can only be true if the hydrogen cyanide concentration was high enough and the rate of reaction of cyanide with ferrous ions was fast enough. We show experimentally that the rate of formation of ferrocyanide is rapid even at low concentrations of hydrogen cyanide in the pH range 6–8, and therefore an equilibrium calculation is valid. The equilibrium concentrations of ferrocyanide are calculated as a function of hydrogen cyanide concentration, pH and temperature. The steady state concentration of hydrogen cyanide depends on the rate of synthesis by electric discharges and ultraviolet light and the rate of hydrolysis, which depends on pH and temperature. Our conclusions show that ferrocyanide was a major species in the prebiotic ocean only at the highest production rates of hydrogen cyanide in a strongly reducing atmosphere and at temperatures of 0°C or less, although small amounts would have been present at lower hydrogen cyanide production rates. The prebiotic application of ferrocyanide as a source of hydrated electrons, as a photochemical replication process, and in semi-permeable membranes is discussed.     

Removal of metal cyanides from aqueous solutions by suspended and immobilized cells of Rhizopus oryzae (MTCC 2541)

This paper presents a study on biodegradation and simultaneous adsorption and biodegradation (SAB) of zinc and iron cyanides by Rhizopus oryzae (MTCC 2541), with a brief process review. Granular activated carbon was used for the immobilization of Rhizopus oryzae (MTCC 2541) for the SAB study. pH and temperature were optimized at an initial cyanide concentration of 100 mg/L for biodegradation and SAB. The microbes adapted to grow at maximum cyanide concentration were harvested and their ability to degrade cyanide was measured in both biodegradation and SAB. The removal efficiency of the SAB process was found to be better as compared to the biodegradation process. In the case of biodegradation, removal was found up to a maximum cyanide concentration of 250 mg CN^?/L for zinc cyanide and 200 mg CN^?/L for iron cyanide, whereas in the case of SAB, about 50% removal of cyanide at 400 mg CN^?/L zinc cyanide and 300 mg CN^–/L iron cyanide was possible. It was found that the SAB process is more effective than biodegradation.     

Conversion of sodium cyanide to carbon dioxide and ammonia by immobilized cells ofPseudomonas putida

Summary Pseudomonas putida, isolated from contaminated industrial wastewaters and soil sites, was found to utilize sodium cyanide (NaCN) as a sole source of carbon and nitrogen. Cells, immobilized in calcium alginate beads (1–2 mm diameter) were aerated in air-uplift-type fluidized batch bioreactor containing 100–400 ppm of NaCN. Degradation of NaCN was monitored for 168 h by analyzing gaseous and dissolved ammonia (NH₃), CO₂, pH and optical density. The results indicated that the alginate-immobilized cells ofP. putida were able to degrade NaCN into NH₃ and CO₂ in a time-dependent manner.     

Transport and metabolism of free cyanide and iron cyanide complexes by willow

Cyanide compounds are contaminants of growing importance that could be remediated biologically via phytoremediation, provided the plants possess suitable mechanisms for managing these pollutants without toxicity. The transport and metabolism of two cyanide compounds, potassium cyanide and potassium ferrocyanide, by willow (Salix eriocephala L. var. Michaux) were compared using a hydroponic system that preserved cyanide speciation and solubility. The cyanide compounds were labelled with ¹⁵N to quantify transport while a novel tissue extraction procedure was used to relate tissue ¹⁵N to cyanide content and speciation. These analyses revealed that although little free cyanide was detected in the aerial tissues of plants exposed to either of these two cyanide compounds, significant enrichments in ¹⁵N were observed, suggesting transport and subsequent metabolism of free cyanide as well as ferrocyanide. The results for ferrocyanide are of interest because this molecule is resistant to microbial degradation and if oxidized to ferricyanide is purportedly membrane impermeable. Nevertheless, these results and mass balance calculations for tissue ¹⁵N and solution cyanide confirming 100% recovery for the added ferrocyanide are suggestive of ferrocyanide uptake and metabolism. This study provides new information describing the biological transport and metabolism of these two cyanide compounds in plants. Moreover, the data also suggest that phytoremediation of cyanide may be possible and ecologically safe due to the lack of cyanide bioaccumulation in aerial tissues.     

Role of transition metal ferrocyanides (II) in chemical evolution

Due to ease of formation of cyanide under prebiotic conditions, cyanide ion might have formed stable complexes with transition metal ions on the primitive earth. In the course of chemical evolution insoluble metal cyano complexes, which settled at the bottom of primeval sea could have formed peptide and metal amino acid complexes through adsorption processes of amino acids onto these metal cyano complexes.Adsorption of amino acids such as glycine, aspartic acid, and histidine on copper ferrocyanide and zinc ferrocyanide have been studied over a wide pH range of 3.6 – 8.5. Amino acids were adsorbed on the metal ferrocyanide complexes for different time periods. The progress of the adsorption was followed spectro-photometrically using ninhydrin reagent. Histidine was found to show maximum adsorption on both the adsorbents at neutral pH. Zinc ferrocyanide exhibits good sorption behaviour for all the three amino acids used in these investigations.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Development of a process for biodetoxification of metal cyanides from waste waters

A bacterial consortium capable of utilising metal cyanides as a source of nitrogen was used to develop a microbiological process for the detoxification of metal cyanides (viz. copper cyanide and zinc cyanide) from electroplating waste water. Optimal conditions biodegradation of both the metal-cyanide compounds were pH 7.5, temperature 35°C, inoculum size 10⁹ cells per ml and glucose or sugarcane molasses requirement of 5 mM or 0.6 ml/l, respectively. Metal precipitates obtained during metal-cyanide biodegradation were identified as metal-hydroxides. When the treatment was carried out in a 27 l rotating biological contactor (RBC) in continuous mode, the system could achieve >99.9% removal of 0.5 mM metal cyanide (ca. 52 mg/l cyanide and 30–40 mg/l copper/zinc) in 15 h with sugarcane molasses as carbon source. The RBC treated effluent was found to be safe for discharge in the environment as confirmed by chemical analysis and fish bioassay studies.     

A spectrophotometric determination of the binding constants of cyanide by hemin in aqueous ethanol solutions

Binding constants for the coordination of cyanide by monomeric hemin in aqueous ethanol solution were determined in the pH range 8–12 by fitting spectrophotometric data to a binding isotherm using a non-linear least-squares method. Two cyanide ligands are coordinated by Fe(III) without evidence for the intermediate mono-ligated species. The binding constants are strongly pH-dependent because of protonation of the ligand and competition for the metal ion by hydroxide. From the pH dependence of the observed binding constants, a pH-independent value of log K = 17.62±0.03 for the binding of two CN⁻ ligands by monomeric hemin in alkaline solution at 25 °C, μ=0.100 M, is obtained.     

Isolation and characterization of a cyanide-utilizing Burkholderia cepacia strain

A bacterium that utilizes cyanide as a nitrogen source was isolated from soil after enrichment in a liquid medium containing potassium cyanide (10mM) and glucose (1.0%, w/v). The strain could tolerate and grow in potassium cyanide at concentrations of up to 25mM. It could also utilize potassium cyanate, potassium thiocyanate, linamarin and a range of aliphatic and aromatic nitriles. The isolate was tentatively identified as Burkholderia cepacia strain C-3. Ammonia and formic acid were found in the culture supernatant of the strain grown on fructose and potassium cyanide, no formamide was detected, suggesting a hydrolytic pathway for the degradation of cyanide. The cyanide-degrading activity was higher in early and the stationary phase cells. Crude cell extracts of strain C-3 grown on nutrient broth exhibited cyanide-degrading activity. The characteristics of strain C-3 suggest that it would be useful in the bioremediation of cyanide-containing waste.     

Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: Role of dark respiration and of light

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 mol m^–2 s^–1. The recovery was partial (< 10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75 mol m^–2 s^–1 did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 mol m^–2 s^–1) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 mol m^–2 s^–1) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - D1- 32 kDa protein of PS II reaction center - FCCP- carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - KCN- potassium cyanide - PBQ- phenyl-p-benzoquinone - PFD- photon flux density - SHAM- salicylhydroxamic acid NBRI Research Publication No. 431.     

Cyanoglycosylation products of 17-O-acetyl-testosterone

17-O-Acetyl testosterone, which has no susceptible hydroxyl or carboxyl group for glycosylation, was glycosylated with 2,3,4,6-tetra-O-acetyl-α- -glucopyranosyl bromide in the presence of a mixed catalyst, Hg(CN)₂ and HgBr₂, in benzene–nitromethane. Reaction occurred on the α,β-unsaturated ketone on the six–membered A-ring to give six 3-O-glycosides, each bearing a cyano group at the 3- or 5-position of the aglycon, and a 3-O-glycoside bearing a CONH₂ group at the 3-position. Structural analyses of these products were carried out by various NMR (¹H, ¹³C NMR, ¹H–¹H and ¹H–¹³C COSY, HMBC, and DEPT), FABMS and X-ray analyses. The mechanisms of the formations of the products are discussed. It was determined that mercuric cyanide was essential as a catalyst for the progress of the cyanoglycosylation.     

Effect of 2,4-Dichlorophenoxyacetic Acid on Endogenous Cyanide, beta-Cyanoalanine Synthase Activity, and Ethylene Evolution in Seedlings of Soybean and Barley

Treatment of etiolated seedlings of barley (Hordeum vulgare) and soybean (Glycine max) with 1 millimolar 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in a 14-fold and greater than 100-fold increase in ethylene production, respectively. Simultaneous monitoring of endogenous cyanide and β-cyanoalanine synthase (β-CAS) (EC 4.4. 1.9) activity was also performed. Endogenous levels of cyanide did not change in barley. In soybean, endogenous cyanide increased within 3 hours, increased again 6 hours after exposure to 2,4-D, and continued to increase throughout the experimental period. The activity of β-CAS increased in both barley and soybean 9 hours after herbicide treatment. The increase in cyanide preceded the increase in β-CAS activity by 3 to 6 hours in soybean. The steady-state concentration of endogenous cyanide in soybean was 1 micromolar, based on rates of ethylene production and cyanide metabolism by β-CAS. This agreed with the determination of endogenous cyanide by both distillation and isotope dilution. Given the apparent compartmentalization of β-CAS in mitochondria and the localization of ethylene/HCN production at the plasmalemma and/or tonoplast, our results suggest that extra-mitochondrial accumulation of cyanide in the cytoplasm may occur. If so, the activity of cyanide-sensitive cytoplasmic enzymes could be adversely affected, thus possibly contributing to the toxicity of 2,4-D.     

Metal poison inhibition of carbonic anhydrase

Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO₂) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley-Liss, Inc.     

Biological cyanide degradation in aerobic fluidized bed reactors: treatment of almond seed wastewater

The continuous aerobic transformation of synthetic cyanide waste-water, amygdalin solutions and almond seed extract containing cyanide was investigated in several fluidized bed reactors. Various inocula consisting of activated sludge or soil slurry were used. Successful inoculation was achieved with simple soil slurry. No significant influence was found between the performance of the systems inoculated with a cyanide contaminated soil and a garden soil. The performance and stability of the reactors with respect to degradation rate were tested for a range of cyanide loading conditions, with feed containing only cyanide, and with different additional carbon sources, as well as various CN ratios at a hydraulic retention time of 24 h. No growth with cyanide as the sole source of carbon and nitrogen was observed. The system with lactate as the organic C-source was capable of operating at cyanide concentrations of 160 ppm cyanide with a conversion rate of 0.125 kg cyanide/m³ d. Ammonia was the end product and the effluent concentration was 0.5 ppm CN^–. The systems with ethanol as the organic C-source could degrade only 0.05 kg cyanide/m³ d, whose feed concentration was 60 ppm cyanide. Amygdalin, an organic cyanide-containing compound present in stone fruit seeds, was fed as a model substrate. Degradation rates up to 1.2 kg COD/m³ d could be measured with no free or organically bound cyanide in the effluent. These rates were limited by oxygen transfer, owing to the large amount of degradable COD. The further investigations with almond seed extracts, confirmed the applicability of the aerobic process to treat food-processing waste streams having low concentrations of cyanide with high COD content.The authors with to thank Dr. Ö.M. Kurt for useful discussions.     

Syntheses, structures, and properties of novel one dimensional copper cyanide coordination polymers

Three new one-dimensional copper coordination polymers have been prepared and fully characterized by single-crystal X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and magnetic susceptibility measurements. The structure of [Cu(CN)₂(bpy)] (1) (bpy = 2,2-bipyridyl) (monoclinic P2₁/c, a = 8.9761(7) Å, b = 16.731(1) Å, c = 8.0224(6) Å, β = 114.437(1)°) consists of Cu(II) metal centers coordinated by three cyanide ligands and chelated by one bpy to form the monomers Cu(CN)₃(bpy) with distorted square pyramidal geometry. Each monomer shares two cyanide ligands with two adjacent monomers to form infinite -Cu(II)-CN-Cu(II)-CN-Cu zigzag chains along the c-axis. The one-dimensional structure of [Cu(CN)(bpy)] (2) (hexagonal P3₂, a = 14.4883(6) Å, b = 12.921(1) Å) is built of tetrahedral Cu(CN)₂bpy metal complexes in which Cu(I) metal centers are coordinated by one nitrogen and one carbon from two different CN ligands, and two nitrogens from one bpy. The two CN ligands act as bridging ligands between adjacent monomers to form helical chains along the 3₂ screw axis. The crystal structure of [Cu₂Cl(CN)(bpy)] (3) (orthorhombic Pbca, a = 17.853(2) Å, b = 6.9724 (9) Å, c = 18.7357 (9) Å) consists of two monomers, CuCl₂(CN) and Cu(bpy)(CN) that share a cyanide ligand to form Cu₂Cl₂(CN)(bpy) dimers. The dimers link to each other by sharing Cl ligands leading to the formation of infinite Cu-Cl-Cu chain decorated by the complex Cu(CN)(bpy). Variable-temperature magnetic measurement shows an overall ferromagnetic behavior for compound 1. The magnetic pathway of compound 1 is through the cyanide bridge connecting apical and equatorial positions of adjacent copper (II) ions.     

Heavy metals (Cu,Zn, Pb,Cd) in sediment,zooplankton and epibenthic invertebrates from the area of the continental slope of the Banc d'Arguin (Mauritania)

The concentrations of copper, zinc, lead and cadmium in the surface sediment (upper 5 mm) were generally higher in the silt fraction than in the bulk sediment. No significant geographical trend in the metal concentrations of the surface sediments was found, nor a correlation between concentrations in bulk sediment as well as in the silt fraction and the % silt could be established. In general, the metal concentrations in both bulk sediment and silt are lower, when compared to marine environments in other climatological regions.In zooplankton, the metal concentrations were relatively high: expressed in µg g^–1 on a dry weight (D.W.) basis, they ranged from 15–90 for copper, 70–580 for zinc, 12–55 for lead and 4–10 for cadmium.In epibenthic invertebrate species, both in crustaceans and bivalve molluscs, the concentrations of copper, zinc, and lead were in the same order of magnitude as compared to corresponding species from other geographical latitudes. Cadmium concentrations were relatively low, ranging from 0.13–0.42 µg g^–1 D.W. in the bivalve molluscs Pitaria tumens and from 0.04–0.27 µg g^–1 D.W. in the shrimp Processa elegantula. Also in the crab species Ilia spinosa, Inachus sp. and Pagurus sp., the cadmium concentrations were low, varying between 0.1 and 0.2 µg g^–1 D.W.No significant relation between the metal concentration in whole-body samples and sediment (either bulk or silt) was present. Also no gradient was apparent in concentrations in organisms sampled at different depths (5 to 200 m) along two off-shore transects perpendicular to the Banc d'Arguin. Data indicated lower metal concentration in epibenthic organisms from sampling stations along a northern transect (southwest of Cap Blanc) than in organisms from the southern transect, off Cap Timiris.Evidence was obtained for a considerable atmospheric input of heavy metals, in particular zinc and lead, in a certain area along the continental slope of the Banc d'Arguin.     

Thiocyanate degradation by Acremonium strictum and inhibition by secondary toxicants

Acremonium strictum, capable of degrading 7.4 g thiocyanate l^–1, was isolated from wastewater condensate from coke-oven gas. Ammonia and sulfate were the final products from thiocyanate degradation with a stoichiometric ratio of near 1:1. The highest degradation activity was at pH 6. Although the degradation rate started to be inhibited above 4 g thiocyanate l^–1, thiocyanate was completely degraded up to 7.4 g l^–1 within 85 h in shake-flask cultures. The degradation of thiocyanate was inhibited by phenol above 625 mg l^–1, by cyanide above 16 mg l^–1, and by nitrite above 100 mg l^–1. However, ammonia and nitrate had negligible inhibition on thiocyanate degradation up to 3 g l^–1 and 1.5 g l^–1, respectively.     

The effect of clays on the oligomerization of HCN

Summary The reaction of 0.1 M HCN and dilute solutions of diaminomaleonitrile (DAMN) at pH 8–9 and 25°C in the presence of suspensions of montmorillonite (bentonite) clays were investigated. Montmorillonite clays inhibit the oligomerization of aqueous solutions of HCN. Yields of colored oligomers, urea, and DAMN, are all diminished by clays, but the rate of loss of cyanide is not significantly decreased. The inhibition of oligomer formation is due to the clay-catalyzed decomposition of DAMN. The absence of strong binding of DAMN to clays was suggested by our failure to detect DAMN when a clay that had been incubated with DAMN was washed with spermidine (6 × 10^–3 g/{ie317-1}). It was established that DAMN does not simply bind to the clays by the observation that the bulk of the radioactivity was recovered from the supernatant in the reaction of¹⁴C-DAMN with montmorillonite. The clay-catalyzed decomposition of DAMN was observed when montmorillonite from two different sources was used and with a variety of homoionic montmorillonites and bentonites. A modification of the established procedure for using the cyanide electrode for cyanide analyses was used to follow the release of HCN from DAMN. This new method can be used in both the acidic and basic pH range and it does not result in the destruction of DAMN by the reagents used for the analysis. Quantitative analyses of the reaction solution from the clay-catalyzed decomposition of DAMN revealed the formation of 1–2 equivalents of HCN per mole of DAMN. The possible significance of these clay-catalyzed reactions in chemical evolution is discussed. Chemical Evolution 35: For the previous papers in this series see Ferris, J P. and Joshi, P.C., (1978), Science 201, 361–362; Ferris, J.P., Narang, R.S., Newton, T.A. and Rao, V.R., (1979), J. Org. Chem. 44, 1273–1278; Ferris, J.P. (1979), Science 203, 1135–1136; Ferris, J.P. and Joshi, P.C., (1979), J. Org. Chem. 44, 2133–2137