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PubMed2006

Mechanism of thio acid/azide amidation.

A combined experimental and computational mechanistic study of amide formation from thio acids and azides is described. The data support two distinct mechanistic pathways dependent on the electronic character of the azide component. Relatively electron-rich azides undergo bimolecular coupling with thiocarboxylates via an anion-accelerated [3+2] cycloaddition to give a thiatriazoline. Highly electron-poor azides couple via bimolecular union of the terminal nitrogen of the azide with sulfur of the thiocarboxylate to give a linear adduct. Cyclization of this intermediate gives a thiatriazoline. Decomposition to amide is found to proceed via retro-[3+2] cycloaddition of the neutral thiatriazoline intermediates. Computational analysis (DFT, 6-31+G(d)) identified pathways by which both classes of azide undergo [3+2] cycloaddition with thio acid to give thiatriazoline intermediates, although these paths are higher in energy than the thiocarboxylate amidations. These studies also establish that the reaction profile of electron-poor azides is attributable to a prior capture mechanism followed by intramolecular acylation.

Acids↗

PubMed1997

The stability of ethanol in human whole blood controls: an interlaboratory evaluation.

Sterile whole human blood control materials were commercially prepared in batches containing anticoagulants and preservatives and approximately 90, 150, and 230 mg/dL ethanol with and without 0.3% (w/v) sodium azide. Aliquots in sealed vials were stored by the manufacturer at 2-8 degrees C until shipped monthly to three academic toxicology laboratories that analyzed them in duplicate by gas chromatographic headspace methods at monthly intervals for one year. The resulting data were pooled, and grand mean values were statistically analyzed to determine the respective alcohol stability in these azide-free and azide-containing blood samples. Azide-containing blood samples showed no alcohol losses during the 1-year period. Azide-free blood containing 1.0% (w/v) sodium fluoride and anticoagulants had small alcohol decreases over time, the total losses after one year being less than 5% of the original alcohol concentrations. The initial alcohol concentration of approximately 40 mg/dL also did not change during storage of additional samples of azide-free blood for one month at 4 degrees C. We concluded that addition of sodium azide to performance-test and control blood specimens for alcohol analysis is unnecessary and unwarranted and that alcohol losses in such blood samples can be minimized by simple appropriate treatments and conditions.

Anticoagulants↗

PubMed1976

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.

Antimycin A↗

PubMed1993

Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.

In previous studies, we have shown that inhibition of oxidative phosphorylation in Clone 9 cells (a nontransformed rat liver cell line) by 5 mM azide results in a marked biphasic stimulation of glucose transport that is mediated by GLUT1 [M. Shetty, J. N. Loeb, and F. Ismail-Beige. Am.J. Physiol. 262 (Cell Physiol. 31): C527-C532, 1992]. The late phase of the response (at 8-24 h) is associated with a doubling of cell GLUT1 content and an 8- to 10-fold increment in GLUT1 mRNA abundance. To investigate the mechanisms mediating GLUT1 mRNA induction, we have examined the effect of incubation in the presence of azide on GLUT1 gene transcription. In nuclear run-on assays, the rate of GLUT1 gene transcription was increased 2.5 +/- 0.3-fold in nuclei from cells exposed to azide for 4 h. Additionally, GLUT1 mRNA turnover was decreased in cells treated with azide: upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with half-lives of 2.3 +/- 0.3 and 8.0 +/- 0.5 h in control cells and cells treated with azide for 4 h, respectively. GLUT1 mRNA half-life was most prolonged (> 12 h) when azide was added subsequent to the addition of actinomycin D, and the half-life continued to be prolonged (6.5 +/- 0.5 h) in cells exposed to azide for 16 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1982

Role of catalase and hydroxyl radicals in the oxidation of methanol by rat liver microsomes.

In view of the presence of adventitious catalase in isolated microsomes, and the requirement for H2O2, it has been suggested that NADPH-dependent oxidation of methanol by rat liver microsomes was mediated exclusively by the peroxidatic activity of catalase. However, H2O2 may also serve as a precursor of the hydroxyl radical, and methanol reacts with hydroxyl radicals to produce formaldehyde. Inhibition of H2O2 production should therefore decrease methanol oxidation by either a hydroxyl radical-dependent pathway or a catalase-dependent pathway. To attempt to clarify some of the controversies concerning the roles of H2O2 and catalase in the microsomal pathway of oxidation of short chain alcohols, studies were carried out to determine the nature of the pathway responsible for methanol oxidation by isolated microsomes. In the absence of the catalase inhibitor azide, methanol may be oxidized primarily by the peroxidatic activity of catalase since there was little effect on methanol oxidation by competing hydroxyl radical scavengers. Azide, which inhibited catalase activity greater than 95%, inhibited NADPH-dependent oxidation of methanol by 30-50%. The azide-insensitive (catalase-independent) pathway of methanol oxidation was inhibited by scavengers of hydroxyl radicals. The inhibition of the scavengers reflected the rate constant for interaction with hydroxyl radicals and was greater at lower concentrations of methanol than at higher concentrations, suggesting competition between the scavengers and methanol. The addition of H2O2 stimulated the oxidation of methanol in the presence of azide; H2O2 may serve as a precursor of the hydroxyl radical. Iron-EDTA, which is known to increase hydroxyl radical production, stimulated the oxidation of methanol in the absence and presence of azide. The stimulation by iron-EDTA was blocked by the competing hydroxyl radical scavengers even in the absence of azide, suggesting that the added iron-EDTA favorably with microsomal catalase for H2O2 to produce hydroxyl radicals (or a species with the oxidizing power of the hydroxyl radical). These results suggest that in microsomes, depending on the absence or presence of azide, methanol may be oxidized by two primary pathways, one involving the peroxidatic activity of catalase, and the other in which hydroxyl radicals, generated from microsomal electron transfer, play a role. In view of the crucial role played by H2O2 in both pathways, inhibition of H2O2 formation should not be interpreted solely as evidence for a role for catalase in the microsomal oxidation of alcohols.

Animals↗

PubMed2006

4'-substituted-4-biphenylyloxenium ions: reactivity and selectivity in aqueous solution.

Azide trapping shows that the 4'-substituted-4-biphenylyloxenium ions 1b-d are generated during hydrolysis of 4-aryl-4-acetoxy-2,5-cyclohexadienones, 2c and 2d, and O-(4-aryl)phenyl-N-methanesulfonylhydroxylamines, 3b and 3c. In addition, the 4'-bromo-substituted ester, 2d, undergoes a kinetically second-order reaction with N3- that accounts for a fraction of the azide adduct, 5d. Since both first-order and second-order azide trapping occurs simultaneously in 2d, the second-order reaction is not enforced by the short lifetime of 1d, which has similar azide/solvent selectivity to the unsubstituted ion, 1a. In contrast the 4'-CN and 4'-NO2 ions 1e and 1f cannot be detected by azide trapping during the hydrolysis of the dichloroacetic acid esters 2e' and 2f' even though 18O labeling experiments show that a fraction of the hydrolysis of both esters occurs through C(alkyl)-O bond cleavage. These esters exhibit only second-order trapping by azide. Correlations of the azide/solvent selectivities of 1a-d with the calculated relative driving force for hydration of the ions (DeltaE of eq 4) determined at the pBP/DN//HF/6-31G and BP/6-31G//HF/6-31G levels of theory suggest that 1e and 1f have lifetimes in the 1-100 ps range. Ions with these short lifetimes are not in diffusional equilibrium with nonsolvent nucleophiles, and must be trapped by such nucleophiles via a preassociation mechanism. The second-order trapping that is observed in these two cases is enforced by the short lifetime of the cations, and may occur by a concerted S(N)2' mechanism or by internal azide trapping of an ion sandwich produced by azide-assisted ionization. Comparison of azide/solvent selectivities of the oxenium ions 1a-c with the corresponding biphenylylnitrenium ions 8a-c shows that 4'-substituent effects on reactivity in both sets of ions are similar in magnitude, although the nitrenium ions are ca. 30-fold more stable in an aqueous environment than the corresponding oxenium ions. The magnitude of the 4'-substituent effects for electron-donating substituents suggest that both sets of ions are more accurately described as 4-aryl-1-imino-2,5-cyclohexadienyl or 4-aryl-1-oxo-2,5-cyclohexadienyl carbocations. Calculated structures of the oxenium ions are also consistent with this interpretation.

Journal Article↗

PubMed1999

Redox control in development and evolution: evidence from colonial hydroids

Redox chemistry, involving the transfer of electrons and hydrogen atoms, is central to energy conversion in respiration, and the control of gene expression by redox state commonly occurs in bacteria, allowing rapid responses to environmental changes, for instance, in the food supply. Colonial metazoans often encrust surfaces over which the food supply varies in time or space; hence, in these organisms, redox control of the development of feeding structures and gastrovascular connections could be similarly adaptive, allowing colonies to adjust the timing and spacing of structures in response to a variable food supply. To investigate the possibility of redox control of colony development, the redox states of hydractiniid hydroid colonies were manipulated experimentally. As in many colonial animals, hydractiniid hydroids display a range of morphological variation from sheet-like forms (i.e. closely spaced polyps with high rates of stolon branching) to runner-like forms (i. e. widely spaced polyps with low rates of stolon branching). In the runner-like Podocoryna carnea, azide, a blocker of the electron transport chain, and dinitrophenol, an uncoupler of oxidative phosphorylation, diminished the largely polyp-driven gastrovascular flow to a similar extent. Measures of the redox state of the polyp epitheliomuscular cells using the fluorescence of NAD(P)H suggest that azide shifts the redox state in the direction of reduction, while dinitrophenol shifts the redox state in the direction of oxidation. Colony development corresponds to redox state in that azide-treated colonies were more runner-like, while dinitrophenol-treated colonies were more sheet-like. Nevertheless, the functional role of polyps in feeding and generating gastrovascular flow probably contributed to a trade-off between polyp number and size such that azide-treated colonies had few large polyps, while dinitrophenol-treated colonies had many small polyps. Regardless of the treatment, P. carnea colonies developed to maturity and produced swimming medusae in the normal fashion. In the sheet-like Hydractinia symbiolongicarpus, treatment with azide resulted in complete suppression of the development of both the stolonal mat and the blastostyles, the reproductive polyps. Azide-treated H. symbiolongicarpus colonies therefore developed in a juvenilized, runner-like manner and much resembled colonies of P. carnea. Following cessation of azide treatment in H. symbiolongicarpus, normal colony development ensued, and both a stolonal mat and blastostyles formed. In both hydroid species, relative oxidization favors sheet-like growth, while relative reduction favors runner-like growth. Since feeding triggers strong contractions of polyp epitheliomuscular cells and results in relative oxidation, this experimental evidence supports the hypothesis of adaptive redox control of colony development and evolution.

Journal Article↗

PubMed1994

ATPase kinetics for wild-type Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the beta-subunit Thr197-->Ser mutation.

Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the Thr197-->Ser mutation in the beta subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high. The Thr197-->Ser mutation in the yeast F1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F1-ATPase for ADP is altered by azide, oxyanions and the Thr197-->Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197-->Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197-->Ser mutation as measured by determining the KiADP values for multisite ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under multisite conditions. Sulfite increases the KiADP values for ATP hydrolysis under multisite conditions approximately threefold for the wild-type and mutant enzymes and the Thr197-->Ser mutation increases KiADP ninefold. The effect of sulfite on KiADP is additive to the effect of the Thr197-->Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197-->Ser mutation on the biochemistry of F1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197-->Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197-->Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP.

Adenosine Diphosphate↗

PubMed1980

Effect of ligands on cytochrome d from Azotobacter vinelandii.

Spectra of oxidized and reduced cytochrome d in particles of A. vinelandii were studied in the presence of the ligands CO, azide, and NH2OH under oxidizing, reducing, and turnover conditions. Under oxidizing conditions, spectral changes were observed on oxidized cytochrome d (absorption maximum at 648 nm) in the presence of CO and NH2OH showing a shift of the maximum to shorter wavelengths (639 and 645 nm, respectively) and a broadening of the half-band width. Under reducing conditions, spectral changes were observed on reduced cytochrome d (absorption maximum at 631 nm) in the presence of CO (absorption maximum at 636 nm), NO, NO2-, and NH2OH (absorption maximum at 642 nm in the presence of dithionite). The spectral changes of cytochrome d in the presence of NH2OH or with dithionite and NO2- were ascribed to the formation of the NO-cytochrome d compound. Under turnover conditions CO, NH2OH, and azide cause a spectral shift of the absorption maximum of cytochrome d from 648 nm to 636, 645, and 655 nm, respectively. With NH2OH and azide a broadening of the half-band width of 7 and 6 nm, respectively, was also observed. The spectral changes caused by CO and NH2OH were interpreted as a binding of the ligands to cytochrome d changing its conformation from the oxidized state absorbing at 648 nm into a more stable liganded form. Since azide does not affect the spectral bands of oxidized and reduced cytochrome d, the spectral change during turnover in the presence of azide were ascribed to a preferential binding of azide to enzymically active conformation of cytochrome d (cytochrome dx).

Azides↗

PubMed2002

Angiotensin protects cortical neurons from hypoxic-induced apoptosis via the angiotensin type 2 receptor.

The effects of angiotensin on mouse cortical neuronal cultures exposed to chemical-induced hypoxia was investigated. Cultures exposed to 10 mM sodium azide for 5 min showed a 17% increase in apoptosis when assayed 24 h postinsult. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 blocked sodium azide-induced cell death suggesting that the NMDA receptor contributes to the mediated cell death. Pretreatment of cultured neurons with angiotensin decreased sodium azide-induced apoptosis by 94%. When the AT(1) receptor was blocked by its receptor antagonist, losartan, angiotensin activation of the AT(2) receptor completely inhibited sodium azide-induced apoptosis. Pretreatment of neurons with the AT(2) receptor antagonist PD123319 resulted in angiotensin reducing sodium azide-induced apoptosis by 48%. These results demonstrate that angiotensin can significantly attenuate sodium azide-induced apoptosis primarily through activation of the AT(2) receptor and suggests that angiotensin may have a protective role in neurons undergoing ischemic injury.

Angiotensin Receptor Antagonists↗

PubMed1979

Photoaffinity labeling of the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1-butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In the absence of activating light, binding studies demonstrate that the o-aryl azide competes for binding with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme is irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide, and the protein loses its capacity for substrate binding in direct proportion to photolabeling. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-3H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sites per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate provided partial protection. Gel isoelectric focusing verified this stoichiometry and indicated that the labeled enzyme has a more acidic isoelectric point than the native enzyme.

Affinity Labels↗

PubMed1996

Proton transport by halorhodopsin.

In halorhodopsin from Natronobacterium pharaonis, a light-driven chloride pump, the chloride binding site also binds azide. When azide is bound at this location the retinal Schiff base transiently deprotonates after photoexcitation with light > 530 nm, like in the light-driven proton pump bacteriorhodopsin. As in the photocycle of bacteriorhodopsin, pyranine detects the release of protons to the bulk. The subsequent reprotonation of the Schiff base is also dependent on azide, but with different kinetics that suggest a shuttling of protons from the surface as described earlier for halorhodopsin from Halobacterium salinarium. This azide-dependent, bacteriorhodopsin-like photocycle results in active electrogenic proton transport in the cytoplasmic to extracellular direction, detected in cell envelope vesicle suspensions both with a potential-sensitive electrode and by measuring light-dependent pH change. We conclude that in halorhodopsin an azide bound to the extracellular side of the Schiff base, and another azide shuttling between the Schiff base and the cytoplasmic surface, fulfill the functions of Asp-85 and Asp-96, respectively, in bacteriorhodopsin. Thus, although halorhodopsin is normally a chloride ion pump, it evidently contains all structural requirements, except an internal proton acceptor and a donor, of a proton pump. This observation complements our earlier finding that when a chloride binding site was created in bacteriorhodopsin through replacement of Asp-85 with a threonine, that protein became a chloride ion pump.

Azides↗

PubMed1996

Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 4. Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases.

Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined. The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1. By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate. These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme. However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose. These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates. The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water. This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.

Acetates↗

PubMed2002

Enhancing reactivity via structural distortion.

To examine how small structural changes influence the reactivity and magnetic properties of biologically relevant metal complexes, the reactivity and magnetic properties of two structurally related five-coordinate Fe(III) thiolate compounds are compared. (Et,Pr)-ligated [Fe(III)(S(2)(Me2)N(3)(Et,Pr))]PF(6) (3) is synthesized via the abstraction of a sulfur from alkyl persulfide ligated [Fe(III)(S(2)(Me2)N(3)(Et,Pr))-S(pers)]PF(6) (2) using PEt(3). (Et,Pr)-3 is structurally related to (Pr,Pr)-ligated [Fe(III)(S(2)(Me2)N(3)(Pr,Pr))]PF(6) (1), a nitrile hydratase model compound previously reported by our group, except it contains one fewer methylene unit in its ligand backbone. Removal of this methylene distorts the geometry, opens a S-Fe-N angle by approximately 10 degrees, alters the magnetic properties by stabilizing the S = 1/2 state relative to the S = 3/2 state, and increases reactivity. Reactivity differences between 3 and 1 were assessed by comparing the thermodynamics and kinetics of azide binding. Azide binds reversibly to both (Et,Pr)-3 and (Pr,Pr)-1 in MeOH solutions. The ambient temperature K(eq) describing the equilibrium between five-coordinate 1 or 3 and azide-bound 1-N(3) or 3-N(3) in MeOH is approximately 10 times larger for the (Et,Pr) system. In CH(2)Cl(2), azide binds approximately 3 times faster to 3 relative to 1, and in MeOH, azide dissociates 1 order of magnitude slower from 3-N(3) relative to 1-N(3). The increased on rates are most likely a consequence of the decreased structural rearrangement required to convert 3 to an approximately octahedral structure, or they reflect differences in the LUMO (vs SOMO) orbital population (i.e., spin-state differences). Dissociation rates from both 3-N(3) and 1-N(3) are much faster than one would expect for low-spin Fe(III). Most likely this is due to the labilizing effect of the thiolate sulfur that is trans to azide in these structures.

Algorithms↗

PubMed2001

Why is there an "inert" metal center in the active site of nitrile hydratase? Reactivity and ligand dissociation from a five-coordinate Co(III) nitrile hydratase model.

To determine how a substitutionally inert metal can play a catalytic role in the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate Co(III) thiolate complex ([Co(III)(S(2)(Me2)N(3)(Pr,Pr))](PF(6)) (1)) that resembles the active site of cobalt containing nitrile hydratase (Co NHase) was prepared. This was screened for reactivity, by using low-temperature electronic absorption spectroscopy, toward a number of biologically relevant "substrates". It was determined 1 will react with azide, thiocyanate, and ammonia, but is unreactive toward nitriles, NO, and butyrate. Substrate-bound 1 has similar spectroscopic and structural properties as [Co(III)(ADIT(2))](PF(6)) (2). Complex 2 is a six-coordinate Co(III) complex containing cis-thiolates and imine nitrogens, and has properties similar to the cobalt center of Co NHase. Substrate binding to 1 is reversible and temperature-dependent, allowing for the determination of the thermodynamic parameters of azide and thiocyanate binding and the rates of ligand dissociation. Azide and thiocyanate bind trans to a thiolate, and with similar entropies and enthalpies (thiocyanate: DeltaH = -7.5 +/- 1.1 kcal/mol, DeltaS = -17.2 +/- 3.2 eu; azide: DeltaH = -6.5 +/- 1.0 kcal/mol, DeltaS = -12.6 +/- 2.4 eu). The rates of azide and thiocyanate displacement from the metal center are also comparable to one another (k(d) = (7.22 +/- 0.04) x 10(-)(1) s(-)(1) for thiocyanate and k(d) = (2.14 +/- 0.50) x 10(-)(2) s(-)(1) for azide), and are considerably faster than one would expect for a low-spin d(6) six-coordinate Co(III) complex. These rates are comparable to those of an analogous Fe(III) complex, demonstrating that Co(III) and Fe(III) react at comparable rates when in this ligand environment. This study therefore indicates that ligand displacement from a low-spin Co(III) center in a ligand environment that resembles NHase is not prohibitively slow so as to disallow catalytic action in nonredox active cobalt metalloenzymes.

Azides↗

PubMed1997

Comparison of the ligand-binding properties of native and copper-less cytochromes bo from Escherichia coli.

The binding of four anionic ligands, cyanide, fluoride, azide and formate, to cytochrome bo purified from Escherichia coli cells grown with a copper supplement (+Cu cyt.bo) is described. Membrane-bound cytochrome bo that lacks the copper component, CuB, of its active site can be prepared from cells grown under conditions where the availability of copper is limited by the presence of a CuI chelator, 2,2'-bicinchinonic acid. The ligand-binding properties of this copper-less enzyme (-Cu cyt.bo) are compared with those of +Cu cyt. bo. As judged from near-UV/visible spectroscopic changes, cyanide forms a low-spin complex with +Cu cyt.bo, whereas azide, fluoride and formate form high-spin complexes. The pH-dependences of binding suggest that for all four of these anionic ligands, both the rates of binding and the binding affinities are primarily dependent on the concentration of their protonated forms. -Cu cyt.bo, which shows less than 15% of the duroquinol oxidase activity of +Cu cyt.bo, binds cyanide, azide and fluoride, but with greatly decreased affinity (<1/30, 1/2000 and 1/2500 respectively at pH5.5 compared with +Cu cyt.bo). The complex of azide with -Cu cyt.bo still seems to be high-spin and azide binding to -Cu cyt.bo is still pH-dependent, although less so than azide binding to +Cu cyt.bo.

Azides↗

PubMed1974

Unimportance of counterflux in the energetics of "downhill" transport.

Adam Kepes suggested that the cellular transport and hydrolysis of orthonitrophenyl-beta-d-galactopyranoside is powered by the counterflux of the d-galactose resulting from beta-galactosidase action within the cell. His explanation would rationalize the unique insensitivity of this galactoside transport to energy poisons such as azide. But contrary to the predictions of this hypothesis, (i) there is no initial large inhibition that progressively lessens as galactose is produced. This was shown with a double wavelength stopped-flow spectrophotometer developed to eliminate interference from turbidity transients. (ii) The azide sensitivity does not increase with an external concentration of galactose sufficient to reverse the thermodynamic gradient. (iii) Mutation in galactose utilization or growth on highly catabolite-repressing regimens did not increase the azide sensitivity, and induction of galactose transport and metabolism did not decrease azide sensitivity. It was found that Kepes measurements must have contained two artifacts. One is that the control rate of hydrolysis decreases with time as the dense cell suspension becomes anaerobic. The other is that azide causes turbidity changes for some time after its introduction. If the former is avoided by magnetic stirring and the latter by double wavelength spectrophotometry or controls without substrate, the inhibition is constant from the earliest time that can be measured. It is therefore concluded that energy-unstarved cells, exposed to azide, still have adequate energy reserves to couple to the downhill transport, although their potential is not adequate to drive accumulation against a concentration gradient.

Azides↗

PubMed1978

Cyanide- and hydroxamate-resistant respiration in Neurospora crassa.

Strain inl-89601 of Neurospora crassa respires exclusively by means of the mitochondrial cytochrome chain. The respiration of this strain is entirely inhibited by cyanide or antimycin A, the classical inhibitors of cytochrome chain respiration. When this strain was grown in the presence of chloramphenicol, however, two additional terminal oxidases were detected. One of these oxidases is inhibited by substituted hydroxamic acids and has been described previously. The second oxidase was not inhibited by cyanide or hydroxamic acid but was inhibited by azide in the presence of both cyanide and hydroxamic acid. This azide-sensitive respiration was due to a single respiratory pathway with a Ki for azide of 200 micrometer. A small amount of azide-sensitive respiration was detected in mitochondrial fractions obtained from chloramphenicol-treated cells, and it is likely that the azide-sensitive oxidase is localized in the mitochondrion. The determinants for the azide-sensitive and hydroxamate-sensitive oxidases segregate in a Mendelian manner in crosses and are either unlinked or not closely linked to each other.

Azides↗