Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts

Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes.     

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.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Changes in endogenous cyanide and 1-aminocyclopropane-1-carboxylic acid levels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves

Leaves of Nicotiana tabacum L. cv. Xanthi necroticum plants form local necrotic lesions at the site of infection by tobacco mosaic virus. During the first seven days post-inoculation, endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-ACC increased in the lesion area. The time course of ACC accumulation coincided with an increase in the endogenous cyanide level which began within two days after inoculation. Concomitantly, the activity of -cyanoalanine synthase, the main HCN detoxifying enzyme, decreased. Likewise, treatment of leaf discs of uninfected plants with ACC led to cyanide accumulation. Exogenously applied KCN caused necrotic spots on tobacco leaves very similar to the whitish centers of virus-induced local lesions. Possible implications of cyanide in cell death during TMV-induced lesion development are discussed.     

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.     

The effect of inorganic phosphate on cyanogenesis by Pseudomonas aeruginosa

The biosynthesis of hydrogen cyanide (HCN) by a strain of Pseudomonas aeruginosa is found to be significantly influenced by inorganic phosphate. Optimum HCN production occurs when the phosphate concentration is between 1 and 10 mM. Above and below this concentration the amount of HCN produced decreases sharply and at 0.1 and 100 mM phosphate low HCN production occurs. If a culture growing at 0.1 mM phosphate and producing low HCN is shifted to 10 mM phosphate, HCN biosynthesis resumes. Experiments with chloramphenicol indicate that de novo-protein synthesis is required for the process.     

Toxicity and degradation of metal-complexed cyanide by a bacterial consortium under sulfate-reducing conditions

Free cyanide at 1 mm decreased the initial sulfate reduction rate of a batch culture of granular sludge from 0.3 to 0.14 mmol d(-1) g(-1) SS (suspended solid), whereas 0.5 mm cyanide had a minimal effect (0.25 mmol d(-1) g(-1) SS). The order of toxicity of metal-complexed cyanides to the sludge was as follows: zinc-complexed cyanide (most toxic) > free cyanide = nickel-complexed cyanide > copper-complexed cyanide (least toxic), which also corresponds well with the order of the stability (dissociation) constants of the metal-cyanide complexes. A consortium degrading cyanide was enriched using nickel cyanide as the sole nitrogen source. This consortium completely removed 0.5 mm of nickel-complexed cyanide under sulfate-reducing conditions in 11 d. Analysis of clone library of 16S rRNA genes shows that the consortium was composed of three major phylotypes including Desulfovibrio.     

Cyanide assimilation in Rhizobium ORS 571: influence of the nitrogenase catalyzed hydrogen production on the efficiency of growth

When cyanide is gradually added to a nitrogenfixing culture, Rhizobium ORS 571 is capable of assimilating large amounts of cyanide using its nitrogenase. Under these conditions the molar growth yield on succinate (Y _succ) increases from 27 at the start of cyanide addition to 38 at the end. The respiratory chain of cells grown at a concentration of 7 mM cyanide is still very sensitive to cyanide. The increase in growth yield is explained by a decrease in hydrogen production by nitrogenase as soon as cyanide is assimilated. This is confirmed by calculating the influence of hydrogen production on Y _succ. Hydrogen production by nitrogenase has a greater influence on growth yields than the presence or absence of hydrogenase activity. At the end of cyanide addition when all cell nitrogen is synthesized from cyanide and no nitrogen fixation occurs, nitrogenase will be in a very oxidized state.     

Enzymes of asparagine synthesis in maize roots

An asparagine synthetase which is active with either glutamine or NH ₄ ⁺ has been found in maize (Zea mays L.) roots. Unlike the enzyme obtained from legume cotyledons, the maize-root enzyme is only slightly more efficient with glutamine (Km, 1.0 mM) than with NH ₄ ⁺ (Km, 2.0–3.0 mM). The activity of this enzyme is higher in the mature root than in the root-tip region, i.e. root cells develop a capacity to make asparagine from glutamine or NH ₄ ⁺ as they mature. -Cyanoalanine synthetase is also present in maize roots. The apparent Km for cysteine is 2.6 mM and for cyanide is 0.57 mM. The enzyme is more active in the root tip than in mature root tissue. Thus, if asparagine were made in the root tip, the cyanide pathway could represent the mechanism of synthesis. It is our contention, however, that this potential is not realized under normal conditions because ¹⁴C-experiments performed previously have indicated a limited availability of both CN and cysteine in the maize root.     

The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide     

Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR

The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, [N82A]CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy. Proton NMR studies of [N82A]CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H₂O vs. D₂O) of the buffer solution. No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.     

Cyanide Metabolism in Higher Plants: Cyanoalanine Hydratase is a NIT4 Homolog

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of β-cyano-l-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called ‘cyanoalanine hydratase’ of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.     

Assay and properties of some sulphur enzymes in coastal sands

Summary Arylsulphohydrolase; cysteine desulphohydrase and thiosulphate cyanide sulphurtransferase were assayed in coastal sands and their properties determined. Low enzyme activity was detected in sands lacking vegetation, but much higher activities were detected in the rhizospheres of climax vegetation;Hippopha? rhamnoides andAmmophila sp. Properties of the enzymes were generally similar to those quoted for the enzymes in soils.     

Hydrogen cyanide release during feeding of generalist and specialist lepidopteran larvae on a cyanogenic plant, Passiflora capsularis

Abstract.  The hydrogen cyanide-based interaction of a strongly cyanogenic plant, Passiflora capsularis , and larvae of two insect herbivores, a generalist ( Spodoptera frugiperda ) and a specialist ( Heliconius erato ), is examined in terms of the combined kinetics of the feeding process and the simultaneous hydrogen cyanide (HCN) liberation, as compared with the natural kinetics of hydrogen cyanide evolution by plant-leaf tissue. There are marked differences in acceptance of P. capsularis by third-instar larvae of specialist and generalist species. The former, H. erato , display a parsimonious ingestion rate of 0.74 ± 0.15 mg (fresh weight) min⁻¹ comprising 18% active feeding time, whereas S. frugiperda larvae show a more erratic and restrained feeding involving 4% of the time at 0.45 ± 0.14 mg min⁻¹. These S. frugiperda larvae ingest 124.4 ± 8.3 mg (fw) of the non-cyanogeneic Spinacia oleracea leaves in 24 h compared with only 74.7 ± 20.1 mg of P. capsularis in the same period. The total hydrogen cyanide released naturally from wild specimens of P. capsularis plants is in the range 326–3901 μg g⁻¹. Hydrogen cyanide evolution from macerated P. capsularis leaves takes place along a hyperbolic function with time and initial velocities of cyanide evolution are a linear function of total hydrogen cyanide. When feeding on P. capsularis leaves, H. erato releases only a minor fraction relative to total hydrogen cyanide (0.09%) and to the anticipated cyanide from the initial velocity (7%). By contrast, S. frugiperda evokes 5.8-fold more than the anticipated hydrogen cyanide release from the plant. The findings are interpreted as diverging strategies by generalist and specialist insects in the utilization of hydrogen cyanide in cyanogenic plants.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

The redox couple of the cytochrome c cyanide complex: the contribution of heme iron ligation to the structural stability, chemical reactivity, and physiological behavior of horse cytochrome c

Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(-1) or 13.4 kJ/mol (3.2 kcal/mol), which is 48.1 kJ/mol (11.5 kcal/mol) less favorable than the binding of cyanide to ferricytochrome c. For coordination of cyanide to ferrocytochrome c, the entropy change was earlier experimentally evaluated as 92.4 J mol(-1) K(-1) (22.1 e.u.) at 25 K, and the enthalpy change for the same net reaction was calculated to be 41.0 kJ/mol (9.8 kcal/mol). By taking these results into account, it was discovered that the major obstacle to cyanide coordination to ferrocytochrome c is enthalpic, due to the greater compactness of the reduced molecule or, alternatively, to a lower rate of conformational fluctuation caused by solvation, electrostatic, and structural factors. The biophysical consequences of the large difference in the stabilities of the closed crevice structures are discussed.     

Utilization of ammonia,generated from abiotic cyanide degradation,by Rhodotorula rubra

The viability of the yeast Rhodotorula rubra, isolated from liquid samples of gold-mine effluents, was not affected by the presence of 11.52 mM cyanide. The yeast was able to utilize ammonia, generated from abiotic cyanide degradation in the presence of reducing sugars, in aerobic culture at pH 9.0. These physiological characteristics encourage studies with mixed cultures of cyanide-degrading organisms, using this yeast as an assimilator of ammonia.The authors are with the Department of Microbiology, Institute of Biological Sciences. Federal University of Minas Gerais, C. P. 486, 31270-901 Belo Horizonte, Brazil     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.     

A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production

Cyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, because of their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A qualitative biochemical screen of the M2 population derived from EMS treatment of sorghum seeds, followed by the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING), was employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterization of these plants identified mutations affecting the function or expression of dhurrin biosynthesis enzymes, and the ability of plants to catabolise dhurrin. The main focus in this study is on acyanogenic or low cyanide releasing lines that contain mutations in CYP79A1, the cytochrome P450 enzyme catalysing the first committed step in dhurrin synthesis. Molecular modelling supports the measured effects on CYP79A1 activity in the mutant lines. Plants harbouring a P414L mutation in CYP79A1 are acyanogenic when homozygous for this mutation and are phenotypically normal, except for slightly slower growth at early seedling stage. Detailed biochemical analyses demonstrate that the enzyme is present in wild-type amounts but is catalytically inactive. Additional mutants capable of producing dhurrin at normal levels in young seedlings but with negligible leaf dhurrin levels in mature plants were also identified. No mutations were detected in the coding sequence of dhurrin biosynthetic genes in this second group of mutants, which are as tall or taller, and leafier than nonmutated lines. These sorghum mutants with reduced or negligible dhurrin content may be ideally suited for forage production.