Nucleophilic addition reactions of free and enzyme-bound deazaflavin.

DeazaFMN-containing glycolate oxidase has been prepared and shown to catalyze the stereospecific transfer of the alpha-hydrogen from substrate to enzyme-bound deazaFMN. The reaction of sulfite, cyanide, and hydroxylamine with several deazaflavin-containing enzymes (glycolate oxidase, D-amino acid oxidase, glucose oxidase, N-methylglutamate synthetase) and free deazaFMN has been examined. All the deazaflavin systems tested form reversible 1:1 complexes with sulfite and cyanide. The pH dependence of the reaction of free deazaFMN with cyanide indicates that cyanide anion is the reacting nucleophile. Hydroxylamine complexes are formed with deazaFMN glycolate oxidase and deazaFAD glucose oxidase. The effectiveness of the various nucleophilic reagents in complex formation decreases in the following order: sulfite greater than cyanide greater than hydroxylamine. The relative stability observed for the sulfite and cyanide complexes formed with various deazaflavin systems (glycolate oxidase greater than D-amino acid oxidase greater than free deazaFMN) follows the same trend observed for the stability of the sulfite complexes formed with the corresponding flavin system. A correlation is also observed between the reduction potential (E'o) of the deazaflavin system (glycolate oxidase (- 170 mV) greater than D-amino acid oxidase (-240 mV) greater than free deazaFMN (-178 mV) and the stability of the deazaflavin-nucleophile complexes. The following evidence indicates that deazaflavin systems are generally more susceptible toward nucleophilic attack than corresponding flavin system: (a) with the exception of glucose oxidase, the dissociation constants for the deazaflavin-sulfite complexes are at least 1 order of magnitude less than the corresponding flavin sulfite complexes; (b) the least reactive nucleophile, hydroxylamine, does not form a complex with any of the flavin systems. In the case of cyanide, a complex is formed only with native glycolate oxidase, which is the flavin-containing system most susceptible to attack by the more reactive sulfite. Formation of the various (deaza)flavin-nucleophile complexes is characterized by a bleaching of the longer wavelength absorption band of the chromophore and increases in absorption below the isosbestic point of the reaction in the near-ultraviolet region of the spectrum. These results are consistent with the formation of covalent adducts via attack of the various nucleophiles at position 5 of (deaza)flavin. The reaction with cyanide provides the first example of a reversible addition of carbanion to enzyme-bound (deaza)flavin.     

Prebiotic Adenine Revisited: Eutectics and Photochemistry

Recent studies support an earlier suggestion that, if adenine was formed prebiotically on the primitive earth, eutectic freezing of hydrogen cyanide solutions is likely to have been important. Here we revisit the suggestion that the synthesis of adenine may have involved the photochemical conversion of the tetramer of hydrogen cyanide in eutectic solution to 4-amino-5-cyano-imidazole. This would make possible a reaction sequence that does not require the presence of free ammonia. It is further suggested that the reaction of cyanoacetylene with cyanate in eutectic solution to give cytosine might have proceeded in parallel with adenine synthesis.     

Methyleneaminoacetonitrile: possible role in chemical evolution-II.

Methyleneaminoacetonitrile (MAAN) when reacted with other amino acids, gives rise to the formation of peptides in addition to the usual hydrolytic products. It acts as a precursor of glycine and also as a dehydration condensing agent. It has been shown that MAAN is easily formed by the reaction of hydrogen cyanide, ammonia and formaldehyde as well as by the reaction of formaldehyde with aminoacetonitrile, in dilute ammoniacal solution.     

Reactions of molybdenum-sulphur compounds with cyanide: chemical evolution and deactivation of molybdoenzymes.

Reactions of molybdenum-sulphur compounds with cyanide are reported which may be relevant to (1) the chemical evolution of molybdoenzymes and (2) deactivation of molybdoenzymes by cyanide. (1) With aqueous cyanide MoS2 gave thio-bridged complex anions [(Mo(CN)6)2(mu-S)]6- and [(Mo(CN)4(mu-S))2]6-. Under prebiotic conditions such complexes could have been formed similarly from molybdenite and may have been precursors of molybdoenzymes. (2) Only those compounds which contained terminal sulphur bound to molybdenum (i.e., Mo = S groups), viz. oxothiomolybdates and the complex [(Mo(mu-S)(S)(Et2NCS2))2], reacted with cyanide; thiocyanate was formed and the molybdenum underwent two-electron reduction. That the cyanolysable sulphur of xanthine oxidase reacts in the same way with cyanide suggests the presence of a Mo = S group which could be a structural feature of the enzyme or could have been formed by initial cyanolysis of a bound persulphide or cysteine residue.     

Archean geochemistry of formaldehyde and cyanide and the oligomerization of cyanohydrin

The sources and speciation of reduced carbon and nitrogen inferred for the early Archean are reviewed in terms of current observations and models, and known chemical reactions. Within this framework hydrogen cyanide and cyanide ion in significant concentration would have been eliminated by reaction with excess formaldehyde to form cyanohydrin (glycolonitrile), and with ferrous ion to form ferrocyanide. Natural reactions of these molecules would under such conditions deserve special consideration in modeling of primordial organochemical processes.As a step in this direction, transformation reactions have been investigated involving glycolonitrile in the presence of water. We find that glycolonitrile, formed from formaldehyde and hydrogen cyanide or cyanide ion, spontaneously cyclodimerizes to 4-amino-2-hydroxymethyloxazole. The crystalline dimer is the major product at low temperature (0 °C); the yield diminishes with increasing temperature at the expense of polymerization and hydrolysis products. Hydrolysis of glycolonitrile and of oxazole yields a number of simpler organic molecules, including ammonia and glycolamide. The spontaneous polymerization of glycolonitrile and its dimer gives rise to soluble, cationic oligomers of as yet unknown structure, and, unless arrested, to a viscous liquid, insoluble in water.A loss of cyanide by reaction with formaldehyde, inferred for the early terrestrial hydrosphere and cryosphere would present a dilemma for hypotheses invoking cyanide and related compounds as concentrated reactants capable of forming biomolecular precursor species. Attempts to escape from its horns may take advantage of the efficient concentration and separation of cyanide as solid ferriferrocyanide, and most directly of reactions of glycolonitrile and its derivatives.     

Possible role of hydrogen cyanide in chemical evolution. The oligomerization and condensation of hydrogen cyanide

The rate of cyanide oligomerization is independent of the presence of added nucleophiles such as azide, monomethylamine or triethylamine and is dependent only on the pH of the reaction mixture. The products formed, with the exception of urea, are also independent of the nucleophiles used to initiate reaction. In the presence of monomethylamine, monomethylurea is the main neutral product instead of urea, suggesting the intermediacy of cyanate. Evidence is presented which suggests that cyanogen may be the precurser to both cyanate and oxalic acid in the cyanide Oligomerization.     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway.

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.     

Formation of cyanomethyl derivatives of basic amino acids and proteins with components in cigarette smoke

A reaction of the basic amino acids, lysine and arginine, with components of cigarette smoke has been observed. The adducts produced have been identified as cyanomethyl derivatives. Both formaldehyde and cyanide, which are known to be present in cigarette smoke, are involved in the reaction with the primary amino group. The reaction is time-dependent and can be enhanced by an increase of temperature or by incubation under alkaline conditions. Cyanomethyl adduct formation was found to be increased when smoke from cigarettes with higher tar and nicotine content was used. When proteins, such as bovine serum albumin, trypsin inhibitors or crude rat lung proteins were incubated with the cigarette smoke solution, new protein adducts with increased pI values were produced which are separable from the original proteins by gel isoelectric focussing. Radioisotopically labelled cyanide can be irreversibly linked to protein and the linkage is enhanced in the presence of formaldehyde.     

Cyanide Degradation under Alkaline Conditions by a Strain of Fusarium solani Isolated from Contaminated Soils

Several cyanide-tolerant microorganisms have been selected from alkaline wastes and soils contaminated with cyanide. Among them, a fungus identified as Fusarium solani IHEM 8026 shows a good potential for cyanide biodegradation under alkaline conditions (pH 9.2 to 10.7). Results of K(sup14)CN biodegradation studies show that fungal metabolism seems to proceed by a two-step hydrolytic mechanism: (i) the first reaction involves the conversion of cyanide to formamide by a cyanide-hydrolyzing enzyme, cyanide hydratase (EC 4.2.1.66); and (ii) the second reaction consists of the conversion of formamide to formate, which is associated with fungal growth. No growth occurred during the first step of cyanide degradation, suggesting that cyanide is toxic to some degree even in cyanide-degrading microorganisms, such as F. solani. The presence of organic nutrients in the medium has a major influence on the occurrence of the second step. Addition of small amounts of yeast extract led to fungal growth, whereas no growth was observed in media containing cyanide as the sole source of carbon and nitrogen. The simple hydrolytic detoxification pathway identified in the present study could be used for the treatment of many industrial alkaline effluents and wastes containing free cyanide without a prior acidification step, thus limiting the risk of cyanhydric acid volatilization; this should be of great interest from an environmental and health point of view.     

Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min⁻¹ ml of culture fluid⁻¹) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by ¹³C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO₂ (and NH₃) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.     

1H, 13C NMR, and electronic absorption spectroscopic studies of the interaction of cyanide with aurothiomalate

The reaction between cyanide and aurothiomalate (Autm) has been studied by 1H and 13C NMR spectroscopy and by uv spectroscopy. At cyanide:Autm ratios greater than or equal to 2, aurocyanide, [Au(CN)2]-, is the sole product but was also produced at lower ratios. Two intermediates were also identified. These were a mixed ligand complex, [tmAuCN]-, which accounted for over 80% of the gold at a ratio of cyanide to Autm of 1, and a bisthiomalato complex, [Autm2]-, which accounted for 6.8% of the total gold at this ratio of cyanide to Autm. The formation of these complexes may be significant in the antiarthritic activity of Autm since cyanide is produced by potential target cells such as polymorphonuclear leukocytes.     

Folding of the polypeptide chain(s), conformational flexibility and reactivity of the metal active site of hemocyanin and tyrosinase

Summary The comparative accessibility of the active sites of hemocyanin and tyrosinase, two proteins containing a binuclear type-3 copper site, has been investigated. The approaches were: (a) the kinetic study of the reaction of hemocyanin with cyanide in the presence of conformation perturbants; (b) the comparison between the kinetic parameters of the cyanide reaction on hemocyanin and tyrosinase; (c) the study of the efficiency and reaction mechanism of hemocyanin interaction with a typical tyrosinase substrate like catechol. The results indicate that the active site of tyrosinase is much more exposed than that of hemocyanin.     

Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min⁻¹ ml of culture fluid⁻¹) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by ¹³C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO₂ (and NH₃) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.Cyanide is a notorious poison. Its inhibitory effect on respiration has been known since the 1920s, when Warburg and Keilin first demonstrated that it combines with trivalent iron in cytochrome oxidase (38, 40, 44). Although highly toxic, it is a normal part of our environment for which mechanisms of biological formation (cyanogenesis) and detoxification exist (8, 22, 42). Cyanide also arises from various industrial practices such as steel coking, electroplating, and mining, but significant accumulations in the environment probably do not occur because of its highly reactive nature (13, 18, 41, 46). The interactions between microorganisms and cyanide, however, remain of interest, since the mechanisms of tolerance and assimilation are poorly understood. A number of reports documenting the ability of microorganisms to grow on cyanide have appeared, but the biochemical basis of these abilities has remained largely obscure. Most studies have reported its ability to serve as a nitrogen source only, since at the concentrations needed for it to serve as a carbon source, it is too toxic (15, 24). As far as is known, growth on cyanide requires that it be enzymatically converted to ammonia. Once formed it can then be readily incorporated into cellular macromolecules by established mechanisms (31). Two separate conversions have been described. They are hydrolytic and oxidative conversion, and they yield formic acid and carbon dioxide as reaction by-products, respectively. The enzyme responsible for hydrolytic conversion has variously been described as cyanidase, cyanide dihydratase, or cyanide nitrilase (CNN), and it catalyzes the reaction shown in equation 1. 1 Mechanistically, CNNs resemble other nitrilases (e.g., EC 3.5.5.1) that catalyze the direct conversion of organic nitriles into an organic acid and ammonia but for which the substrate range appears to be limited to cyanide. The involvement of CNNs in cyanide metabolism has been reported for Alcaligenes xylosooxidans subsp. denitrificans (19, 20), Bacillus pumilus (30), and Pseudomonas sp. (45). Oxidative conversion is mediated by an enzyme described as cyanide oxygenase (CNO). This enzyme has been described for Pseudomonas fluorescens NCIMB 11764 only (15, 23–26). Recent work in our laboratory has shown that CNO functions as a monooxygenase, since a single atom of molecular oxygen was shown to be incorporated during conversion (43). Since the other atom of oxygen in CO₂ was shown to be derived from water, a reaction mechanism in which cyanide undergoes initial monooxygenative attack to give an unknown intermediate (X-OH) as shown in equation 2 was proposed (43). 2 Further hydrolysis of X-OH is then expected to give CO₂ and NH₃ as shown in equation 3. 3 The nature of X-OH and whether an additional enzyme is required for its conversion are unknown. Interestingly, NCIMB 11764 also elaborates a CNN, but only CNO has been shown to be physiologically required for cyanide utilization (26). This conclusion was reached after it was found that mutants unable to grow on cyanide did not make CNO but could still elaborate CNN.CNO is induced when cyanide (KCN) is added to nitrogen-limited cells (4, 26). This approach for obtaining cells induced for the enzyme is more convenient than growing cells on cyanide, which requires several days of fed-batch cultivation. During the course of experiments aimed at optimizing CNO induction, we discovered that the consumption of cyanide and the appearance of CNO activity in cell extracts were not concomitant (3, 4). Further experiments showed that cyanide consumption independent of that catalyzed by CNO occurred nonenzymatically and that a reaction between cyanide and a metabolite excreted into the medium was responsible for cyanide’s removal (4). Since cyanide-removing activity in culture fluids consistently copurified with iron-chelating activity, it was concluded that the responsible metabolite was a siderophore, but further identification of this siderophore was not achieved. Here we report that the compounds responsible for nonenzymatic cyanide removal are α-keto acids, namely, pyruvate (Pyr) and α-ketoglutarate (αKg). These findings help explain the earlier reported involvement of a putative siderophore, since these compounds can act as iron chelators (10, 35). However, the additional ability to serve also as effective cyanide-scavenging agents has not generally been recognized. Both Pyr and αKg were excreted into the medium when P. fluorescens NCIMB 11764 was grown on nitrogen-limiting amounts of ammonia or cyanide as a nitrogen source, and we now demonstrate that these metabolites play an essential role in the utilization of cyanide as a growth substrate.     

Enzymatic assimilation of cyanide via pterin-dependent oxygenolytic cleavage to ammonia and formate in Pseudomonas fluorescens NCIMB 11764

Utilization of cyanide as a nitrogen source by Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, with the latter compound satisfying the nitrogen requirement. Substrate attack is initiated by cyanide oxygenase (CNO), which has been shown previously to have properties of a pterin-dependent hydroxylase. CNO was purified 71-fold and catalyzed the quantitative conversion of cyanide supplied at micromolar concentrations (10 to 50 micro M) to formate and ammonia. The specific activity of the partially purified enzyme was approximately 500 mU/mg of protein. The pterin requirement for activity could be satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 micro M). These compounds included, for example, biopterin, monapterin, and neopterin, all of which were also identified in cell extracts. Substrate conversion was accompanied by the consumption of 1 and 2 molar equivalents of molecular oxygen and NADH, respectively. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted and displayed an overall reaction stoichiometry of 1:1:1 for cyanide, O(2), and NADH consumed. Cyanide was also attacked by CNO at a higher concentration (1 mM), but in this case formamide accumulated as the major reaction product (formamide/formate ratio, 0.6:0.3) and was not further degraded. A complex reaction mechanism involving the production of isocyanate as a potential CNO monooxygenation product is proposed. Subsequent reduction of isocyanate to formamide, whose hydrolysis occurs as a CNO-bound intermediate, is further envisioned. To our knowledge, this is the first report of enzymatic conversion of cyanide to formate and ammonia by a pterin-dependent oxygenative mechanism.     

Abiotic synthesis of purines and other heterocyclic compounds by the action of electrical discharges

Summary The synthesis of purines and pyrimidines using Oparin-Urey-type primitive Earth atmospheres has been demonstrated by reacting methane, ethane, and ammonia in electrical discharges. Adenine, guanine, 4-aminoimidazole-5-carboxamide (AICA), and isocytosine have been identified by UV spectrometry and paper chromatography as the products of the reaction. The total yields of the identified heterocyclic compounds are 0.0023%. It is concluded that adenine synthesis occurs at a much lower concentration of hydrogen cyanide than has been shown by earlier studies. Pathways for the synthesis of purines from hydrogen cyanide are discussed, and a comparison of the heterocyclic compounds that have been identified in meteorites and in prebiotic reactions is presented.     

A facile one-pot synthesis of 4,5-diaryl-2,2-dimethyl-3(2H)-furanones

An efficient and practical one-pot synthesis of 4,5-diaryl-2,2-dimethyl-3(2H)-furanones has been achieved from 1,2-diarylethanones and 2-bromoisobutyryl cyanide in the presence of excess base, by employing the 'hard soft acid base' principle. The reaction scope of 2-bromoisobutyryl cyanide could be expanded to prepare a variety of 2,2-dimethyl-3(2H)-furanone derivatives other than 4,5-diaryl-2,2-dimethyl-3(2H)-furanones.     

The effect of intermediate respiratory chain inhibitors upon the tetrazolium-reductase activity of human polymorphonuclear leukocytes.

Intact human phagocytes are capable of reducing tetrazolium salts in their cytoplasm. The exact mechanism that reduces the dye is not well understood. It has been suggested that increased NADH and stimulation of NADH-oxidase may be responsible for tetrazolium reduction. This reaction is insensitive to terminal respiratory inhibitors such as cyanide, but the effect of intermediate respiratory inhibitors has not been explored. Amytal and Antimycin A were used in blood from 60 healthy subjects to study its effect upon the biochemical and histochemical tetrazolium-reductase reactions. It was found that Amytal and Antimycin block the reaction, while azide and cyanide have no effect. It was concluded that electrons are trapped between cytochromes b and C₁ and that this portion of the respiratory chain is essential for tetrazolium reduction. Since CoQ is the main constituent of that portion of the chain, we assume that this compound could be of prime importance in the biochemical events that lead to intracytoplasmic tetrazolium reduction. The possibility of CoQ participation in some phagocytic functions and in phagocytic disorders is discussed.     

A bridged Mo(VI) complex containing six and five coordinate Mo: Synthesis, 95Mo N.m.r. spectroscopy and X-ray crystal structure of Mo2O5(C17H17N2O)2

The kinetics of the reaction between Carcinus maenas hemocyanin and cyanide has been studied at various KCN concentrations and a different temperatures (21° and 4°C) by following the decrease of the copper-peroxide absorption band, centered at 337 nm, of the copper still bound to the protein and the intrinsic fluorescence changes as functions of time. In all conditions used, the absorption band completely disappears and KCN concentration affects only the rate of the process. The reaction is kinetically homogeneous indicating no site-site interaction. The apparent rate constant increases with the square of cyanide concentration and the inverse of O₂ concentration. The copper still bound decreases at a rate slower than the 337 nm absorption and the process is not kinetically homogeneous. The fluorescence of the protein increases after an induction period showing an inflection point at about 50% of the total effect. A kinetic model has been proposed on the assumption that the two metal ions are removed sequentially from the active site. The experimental data are in agreement with the theoretical equations derived from the model. The equilibrium constants for the formation of the complex between the first and the second copper ion with cyanide and the rate constants of their decomposition have been calculated. The rate-limiting process for the removal of the second copper ion is the formation of the complex with cyanide.     

Cyanide and azide behave in a similar fashion versus cuprozinc-superoxide dismutase

The 1H NMR spectra of the cyanide adduct of Cu2Co2-superoxide dismutase have been remeasured at pH 7.5. The exchange rate of CN- is slow on the NMR time scale. The correlation with the spectrum of the unligated enzyme has been established through saturation-transfer techniques of the system in which 50% of the cyanide adduct is formed and through comparison with the spectrum of a Cu2Co2-superoxide dismutase-CN- sample in which the histidines have been deuterium labeled at the position epsilon 1. The similarities between the spectra of the CN- and N-3 derivatives are stressed, in particular with respect to the removal from copper coordination of the same histidine, assigned as His-46.