Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

Electron spin resonance investigation of the cyanyl and azidyl radical formation by cytochrome c oxidase.

Cyanide (CN(-)) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a(3) of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ((.)CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(.)CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N(3)(-)). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/(.)N(3) radical adduct was readily detected when H(2)O(2) was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

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.     

Acceptor substrate-potentiated inactivation of bovine liver rhodanese

The interaction of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with the acceptor substrates, dithiothreitol or cyanide, was studied. When incubated in the presence of cyanide or dithiothreitol, rhodanese was inactivated in a time-dependent process. This inactivation was detectable only at low enzyme concentrations; the rate and degree of inactivation could be modulated by varying the substrate concentration or the system pH. Activity measurements and fluorescence spectroscopy techniques were used in examining the inactivation phenomenon. Sulfur transfer to dithiothreitol was measured by direct assay and was shown to involve the dequenching of enzymic intrinsic fluorescence that had been previously observed only with cyanide as the acceptor substrate. Substrate-potentiated inactivation of rhodanese (with cyanide) has been reported before, but the cause and nature of this interaction were unexplained. The results presented here are consistent with an explanation invoking oxidation of rhodanese in the course of inactivation.     

Indications to an NADPH oxidase as a possible pO2 sensor in the rat carotid body

The rat carotid body superfused with low pO2 exhibited an optical absorbance spectrum which resembles the reduced spectrum of the NADPH oxidase in neutrophils. Diphenylene iodonium (DPI) as a specific inhibitor of the oxidase attenuated the reduced absorbance spectrum in the carotid body. Also absorbance bleaching by low doses of cyanide (50 and 100 microM) was inhibited by DPI, whereas higher doses of cyanide (300 microM) caused an absorbance spectrum typical for reduced cytochromes. It is concluded that an NADPH oxidase acts as a pO2 sensor in the carotid body with low affinity for oxygen and high affinity for cyanide.     

Cyanide Resistance in Achromobacter II. Mechanism of Cyanide Resistance.

Oka, Tetuo (University of Tokyo, Tokyo, Japan), and Kei Arima. Cyanide resistance in Achromobacter. II. Mechanism of cyanide resistance. J. Bacteriol. 90:744-747. 1965.-Photochemical data showed that the only oxidase found in the cyanide-sensitive cells of Achromobacter was cytochrome o, and that cyanide-resistant cells contained at least two oxidases. The oxidase responsible for cyanide resistance was a pigment the CO compound of which had its absorption band at a wavelength longer than 580 mmu. In addition, kinetic data suggested that there were two oxidases having different affinities for cyanide. From the data presented, resistance to cyanide in Achromobacter strain D was attributed to the induced formation of cytochrome a(2), which has a very low affinity for cyanide. Several characteristics of cytochrome a(2) as a cytochrome oxidase are summarized.     

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.     

ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets

Several free radical intermediates formed during synthesis of prostaglandin H synthase (PGHS) catalyze the biosynthesis of prostaglandins from arachidonic acid (AA). We attempted to directly detect free radical intermediates of PGHS in cells. Studies were carried out using human platelets, which possess significant PGHS activity. Electron spin resonance (ESR) spectra showed a g = 2.005 signal radical, which was formed by the incubation of collagen, thrombin, AA, and a variety of peroxides with human platelets. The ESR spectra obtained using 5,5-dimethyl-1 pyrroline N-oxide (DMPO) and alpha-phenyl N-tert.-butylnitron (PBN) were typical of an immobilized nitroxide. Extensive Pronase digestion of both the DMPO and PBN adducts allowed us to deduce that it was a carbon-centered radical. The formation of this radical was inhibited by potassium cyanide and by desferroxamine. Peroxides stimulated formation of the g = 2.005 signal radical and inhibited platelet aggregation induced by AA. PGHS cosubstrates increased the intensity of the radical signal but inhibited platelet aggregation induced by AA. Both S-nitro-L-glutathione and reduced glutathione quenched the g = 2.005 radical but could not restore platelet aggregatory activity. These results suggest that the carbon-centered radical is a self-destructing free radical formed during peroxide-mediated deactivation of PGHS in human platelets.     

氰化物污染的植物修复可行性研究

氰化物是目前世界范围内最常使用的提取黄金和白银等贵重金属的沥滤剂,其对自然生态环境的污染和破坏以及对人畜和其它生物的毒性作用是众所周知的.本试验用一自行设计的生物反应器来观察黄豆(Glycine max(L)Merr.)和玉米(Zea mays L.)对氰化物污染土壤的原位修复的可能性.室温条件下(23.0～26.0℃),低浓度的氰化物污染液对(≤45.5 CN mg·L^-1)二种测试植物的生长没有产生任何毒性作用;而在高浓度的氰化物试验组(≥91.0 CNmg·L^-1),二种测试植物的生长都出现了明显的滞长现象(生长率下降大于10%),但没有观察到其它毒性反应.同时二种测试植物的叶片细胞用来测定植物细胞线粒体中的氰丙氨酸合成酶(β-cyanoalanine synthase)转化氰化物的潜力.实验是在一封闭的玻璃器皿(100mL)中进行的(100mL的氰化钾溶液中加入1.5g(鲜重)植物的叶片,氰化钾溶液的浓度大约1.0 CNmg·L^-1).在为期28 h的时间内,水溶液中超过90%的氰化物被植物的叶片去除;黄豆和玉米的的叶片细胞对氰化物去除率分别测定为4.43mg CN·kg^-1(鲜重)·h^-1和3.42mg CN·kg^-1(鲜重)·h^-1.本实验结果表明,植物对氰化物污染的土壤原位修复方法是一种可行的和有效的选择.     

Experimentally induced chromosome aberrations in plants. I. The production of chromosome aberrations by cyanide and other heavy metal complexing agents

The finding of Lilly and Thoday that potassium cyanide produces structural chromosome changes in root tips of Vicia faba was confirmed. Like mustards, diepoxides, and maleic hydrazide, potassium cyanide seems to act on cells at early interphase. A tendency of cyanide breaks to be concentrated in heterochromatic segments of the chromosomes was evident. The production of chromosome aberrations by cyanide proved to be practically unaffected by the temperature during treatment. In agreement with Lilly and Thoday, the effect of potassium cyanide was found to be dependent on oxygen tension during treatment. The effect of potassium cyanide increases with increasing oxygen concentration up to 100 per cent oxygen. In the absence of oxygen, potassium cyanide was not completely inactive, but produced a low, though significant frequency of aberrations. Pretreatments with 2.4-dinitrophenol did not influence the effect of potassium cyanide. When bean roots were treated with potassium cyanide before a treatment with 8-ethoxycaffeine, or at the same time as they were treated with 8-ethoxycaffeine, the effect of 8-ethoxycaffeine was almost completely suppressed. The effects of a number of other heavy metal complexing agents were also tested. Sodium fluoride, potassium thiocyanate, carbon monoxide, o-phenanthroline, 2.2-bipyridine, and sodium azide were without radiomimetic effect under the conditions employed, and so was a mixture of sodium azide and sodium fluoride. A low, but quite significant, radiomimetic effect was obtained after treatments with sodium diethyldithiocarbamate, cupferron, and 8-hydroxyquinoline. Under anaerobic conditions, the effects of cyanide and cupferron were both quantitatively and qualitatively indistinguishable. Unlike the effect of cyanide, the effect of cupferron was not enhanced by the presence of oxygen. The effects of the same heavy metal complexing agents were tested on the activities of the enzymes catalase and peroxidase. The activities of both of these enzymes were found to be totally inhibited only by potassium cyanide. In the other cases, little correlation was found between ability to inhibit the activities of these enzymes and ability to produce chromosome aberrations. In a number of experiments, hydrogen peroxide was found to be without radiomimetic effect, whether alone or in combination with potassium cyanide. t-Butyl hydroperoxide proved to be active. The effect of t-butyl hydroperoxide was substantially increased by pretreatments with 2.4.-dinitrophenol. The results are discussed, and it is concluded that the observations made do not support the hypothesis that hydrogen peroxide is involved in the production of chromosome aberrations by potassium cyanide. The possibility that organic peroxides are involved cannot be excluded on the bases of the experimental results. As an alternative hypothesis, it is suggested that iron or other heavy metals are present in the chromosomes and that cyanide and other heavy metal complexing agents produce chromosome aberrations by reacting with these metals.     

Inhibition of aerobic respiration and dissimilatory perchlorate reduction using cyanide

The effect of low concentrations of cyanide on dissimilatory perchlorate and chlorate reduction and aerobic respiration was examined using pure cultures of Azospira sp. KJ. Cyanide at a concentration of 38 microM inhibited cell growth on perchlorate, chlorate and molecular oxygen, but it did not inhibit the activity of chlorite dismutase. When oxygen accumulation was prevented by adding an oxygen scavenger (Oxyrase or L-cysteine), however, cells completely reduced perchlorate in the presence of cyanide. It was concluded that the inhibition of dissimilative perchlorate reduction by cyanide at this concentration was a consequence of oxygen accumulation, not inhibition of the enzymes used for perchlorate reduction. This finding on the effect of cyanide on respiratory enzymes provides a new method to control and study respiratory enzymes used for perchlorate reduction.     

ENDOR from nitrogens and protons in low spin ferric heme and hemoprotein.

Electron nuclear double resonance (ENDOR) signals have been obtained from iron-linked nitrogens in frozen solutions of cytochrome c, metmyoglobin cyanide, and a low spin protohemin mercaptide complex. Hyperfine couplings from heme protons have also been obtained from metmyoglobin cyanide and from a low spin protohemin cyanide complex. Several of these proton resonances are assigned to specific heme protons.     

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.     

Modification by cyanide of the aniline-binding reaction with cytochrome P-450.

Hydroxylation of aniline to p-aminophenol catalyzed by the cytochrome P-450-containing monooxygenase system of liver microsomes is inhibited by cyanide, but microsomal NADPH-cytochrome c reductase is insensitive to this inhibitor. The interaction of aniline with membrane-bound cytochrome P-450, according to spectrophotometric analyses, consists of two phases with respect to aniline concentration, and cyanide interferes differently with these two reaction phases. Noncompetitive and competitive (or mixed type) inhibitions of the aniline-binding reaction by cyanide are observed in reaction systems containing low and, high concentrations of aniline, respectively, a situation similar to the inhibitory action of cyanide on aniline hydroxylase activity. Abnormal aniline-induced difference spectra appeared when cyanide was added as the spectral modifier, and the magnitude of the spectral change in the presence of both aniline and cyanide was a nonadditive change. These results suggest the dissociation of the cytochrome P-450·cyanide complex by aniline. A similar result indicating dissociation of the complex was also obtained by epr spectroscopy. We therefore suggest that addition of a high concentration of substrate causes insensitivity of the microsomal hydroxylase system to cyanide.     

Investigations of cyanide as an infrared probe of hemeprotein ligand binding sites

The measurement of infrared spectra for cyanide liganded to hemeproteins and hemins has been investigated. The hemeproteins included human methemoglobin A, lamprey methemoglobin, metchlorocruorin, horse metmyoglobin, and horseradish peroxidase. The hemins were dicyanide and monopyridine monocyanide species of deuteroporphyrin IX iron(III) and its 2,4-divinyl(proto) and 2,4-diacetyl derivatives. C-N stretch bands of low intensity detected near 2100 cm-1 exhibit changes in frequency, width, intensity, and isotope shift with changes in cyanide compound structure. Infrared band parameters are particularly sensitive to a change in oxidation state (Fe2+ versus Fe3+) and are affected to a lesser extent by changes in porphyrin ring substituent, ligand trans to the cyanide, and protein structure. Evidence of multiple conformers (i.e. multiple C-N stretch bands) was found for several hemeproteins. The cyanide infrared spectra provide direct evidence for cyanide binding as a metal cyanide (Fe--C identical to N) and against HCN being the ligand in nitrile-like bonding (Fe--N identical to C--H) in all the hemeprotein and hemin cyanides studied. With the reduced horseradish peroxidase cyanide, differences between infrared spectra for D2O and H2O solutions can result from hydrogen bonding between a protein amino acid residue and the distal atom of the cyanide (Fe--C identical to N...H+--R). The binding of cyanide to reduced iron (Fe2+) of a hemeprotein was only observed in the case of the reduced peroxidase. These findings demonstrate that cyanide infrared spectra can not only determine when cyanide is bound to a metalloprotein but can also provide information on how the cyanide is bonded to metal and on characteristics of the ligand binding site.     

Correlation analysis between some blood properties and atmospheric environmental parameters

S.W. Tromp made investigations of a weather effect on erythrocyte sedimentation rates (ESR) of human blood by routine checks of the blood of donor groups in Leiden from 1955 to 1985. A higher ESR was found for the summer season and lower ESR for winter and low values occurred soon after a strong cooling spell. In this report, we have continued his work using data for the years 1971–1985 from Leiden (The Netherlands). An influence of the weather on ESR was also found, but this seems to be more complicated than Tromp supposed. Some new aspects are described and elaborated, e.g. ESR already increases before warming is noticed at the ground layer. This is possibly caused by changes in the upper atmosphere, a suggestion that requires further studies. For periods of low ESR, there was a greater number of occlusion fronts passing the Netherlands. The long-term fluctuations of ESR that were found were correlated with sun spot relative numbers only in a few periods. The results of our study justify further research for a variety of other locations around the world.     

Methods for assaying cyanide in bacterial culture supernatant

AIMS: To find an easy, rapid and direct method for the quantitation of cyanide in a moderate number of bacterial culture supernatants. METHODS AND RESULTS: Culture supernatant from stationary phase cultures of Pseudomonas aeruginosa, grown in LB media, were analysed for cyanide content using the Merckoquant and Spectroquant cyanide detection kits as well as a cyanide ion-selective electrode (ISE) and a cyanide micro-ISE. The Merckoquant kit, designed for detection of low quantities of cyanide in water systems, proved not to be sufficiently reliable, providing poor comparison with previous assessments of cyanide levels in Ps. aeruginosa. The Spectroquant kit, and the two ISEs all provided very similar results, in agreement with previous data; however, it was the ISEs that fulfilled all the criteria for a rapid, direct test in a moderate number of samples. CONCLUSIONS: Cyanide ISEs can be used for easy assessment of the cyanide quantity in cultures grown in LB medium. Significance and Impact of the Study: The use of a cyanide ISE allows for an easy, direct and reproducible method for assaying cyanide in bacterial culture supernatant, which is of significant advantage over the currently accepted methods. This is especially important in an era of high-output genomic studies for assessing the phenotypic significance of data relating to the cyanide synthetic genes.     

Kinetics of enzymatic degradation of cyanide

CYANIDASE(@) is a new enzyme preparation capable of degrading cyanide in industrial wastewaters to ammonia and formate in an apparently one-step reaction, down to very low concentrations. This enzyme has both a high selectivity and affinity toward cyanide. A granular form of the biocatalyst was used in a recirculation fixed bed reactor in order to characterize the new biocatalyst with respect to pH, ionic strength, common ions normally present in wastewaters, mass transfer effects, and temperature. Long term stability was investigated. The kinetics of the enzymatic degradation of cyanide were studied in a batch reactor using the powdered immobilized enzyme preparation and modeled using a simple Michaelis-Menten equation.     

Achromobacter cycloclastes nitrite reductase. The function of copper, amino acid composition, and ESR spectra.

1. Dialysis against cyanide at pH 7 of Achromobacter cycloclastes nitrite reductase [EC 1.7.99.3] of a dissimilatory type led to the removal of about 50% of the copper from the enzyme molecule, with a concomitant decrease of the enzymatic activities. It was inferred that enzyme-bound copper atoms play an essential role in the catalytic activities of the enzyme. 2. The amino acid composition of the enzyme was determined after acid hydrolysis. 3. ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper: Type 1 Cu2+, which had narrow and sharp hyperfine splitting, and Type 2 Cu2+, which had broader hyperfine splitting. The bond between the oxidized enzyme and nitrite seems to be ionic.