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PubMed1982

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I.

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.

Chloride Peroxidase↗

PubMed2003

Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant, alpha-bromo fatty aldehyde.

Plasmalogens are a subclass of glycerophospholipids that are enriched in the plasma membrane of many mammalian cells. The vinyl ether bond of plasmalogens renders them susceptible to oxidation. Accordingly, it was hypothesized that reactive brominating species, a unique oxidant formed at the sites of eosinophil activation, such as in asthma, might selectively target plasmalogens for oxidation. Here we show that reactive brominating species produced by the eosinophil peroxidase system of activated eosinophils attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by eosinophil peroxidase target the vinyl ether bond of plasmalogens resulting in the production of a neutral lipid and lysophosphatidylcholine. Chromatographic and mass spectrometric analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by eosinophil peroxidase attacked the plasmalogen vinyl ether bond at acidic pH. Bromide was the preferred substrate for eosinophil peroxidase, and chloride was not appreciably used even at a 1000-fold molar excess. Furthermore, 2-BrHDA production elicited by eosinophil peroxidase-derived reactive brominating species in the presence of 100 microM NaBr doubled with the addition of 100 mM NaCl. The potential physiological significance of this pathway was suggested by the demonstration that 2-BrHDA was produced by phorbol myristate acetate-stimulated eosinophils and by the demonstration that 2-BrHDA is a phagocyte chemoattractant. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by eosinophil peroxidase and by activated eosinophils, resulting in the production of brominated fatty aldehydes.

Acetaldehyde↗

PubMed1983

Steady state kinetics of chloroperoxidase-catalyzed N-demethylation reactions.

The kinetic mechanism of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was determined from initial rate and inhibition studies. When the concentration of N,N-dimethylaniline was varied in the presence of several different fixed concentrations of ethyl hydroperoxide, a double reciprocal plot of the data gave a series of parallel lines. Parallel lines were also observed when the data were plotted as a function of the concentration of ethyl hydroperoxide. A linear double reciprocal plot was obtained when the concentrations of both substrates were varied in a constant ratio. Competitive substrate inhibition was observed for N,N-dimethylaniline with a KI of 4.78 mM. Competitive substrate inhibition was also observed for ethyl hydroperoxide but the secondary replot of the slopes of the double reciprocal lines versus the concentrations of ethyl hydroperoxide intercepted the origin, indicating that no abortive binary complex between ethyl hydroperoxide and the enzyme was formed. These results suggested that the inhibition of the demethylation reaction by ethyl hydroperoxide was due to the reaction of ethyl hydroperoxide with chloroperoxidase compound I to evolve oxygen. N,N-Dimethylaniline inhibited the chloroperoxidase-catalyzed evolution of oxygen from ethyl hydroperoxide with a KI (0.111 mM) essentially identical with its KM for demethylation (0.122 mM). The inhibition of the demethylation reaction by 2,5-dimethylfuran was competitive with respect to N,N-dimethylaniline and uncompetitive with respect to ethyl hydroperoxide, consistent with dead-end inhibition in a ping-pong system. The results of the initial rate and inhibition studies are consistent with a Ping Pong Bi Bi mechanism as the minimal kinetic model for chloroperoxidase-catalyzed N-demethylation reactions. In this model, ethyl hydroperoxide reacts with chloroperoxidase to form the oxidized enzyme intermediate compound I with the concomitant release of ethanol. N,N-Dimethylaniline then binds to compound I and is oxidized, resulting in the formation of N-methylaniline and formaldehyde and the regeneration of the native peroxidase.

Acetaldehyde↗

PubMed1983

The oxidation of 2-aminofluorene by prostaglandin endoperoxide synthetase. Comparison with other peroxidases.

We have examined the prostaglandin endoperoxide synthetase-dependent metabolism of the arylamine carcinogen 2-aminofluorene (2-AF). Ram seminal vesicle microsomes fortified with arachidonic acid metabolize 2-AF to products covalently bound to microsomal macromolecules, water-soluble metabolites, and two organic extractable metabolites. The organic extractable metabolites were identified by co-chromatography, uv-visible spectrophotometry, and mass spectrometry as 2-nitrofluorene and 2,2'-azobisfluorene (azofluorene). Hydrogen peroxide also supports 2-AF metabolism to the same products, suggesting that the hydroperoxidase activity of prostaglandin endoperoxide synthetase is responsible for the co-oxidation. The highly reactive oxygenated metabolites of 2-AF, N-hydroxy-2-AF, and 2-nitrosofluorene, are metabolized by prostaglandin endoperoxide synthetase to one major product, 2-nitrofluorene. The metabolism of 2-AF, N-hydroxy-2-AF, and 2-nitrosofluorene is extremely rapid, reaching completion in less than 30 s. The horseradish peroxidase/H2O2 system also metabolites 2-AF to 2-nitrofluorene and azofluorene. The chloroperoxidase/H2O2 system, however, yields primarily 2-nitrosofluorene from 2-AF. These results suggest that 2-AF is oxidized to an electrophilic intermediate(s) by prostaglandin endoperoxide synthetase, which either binds covalently to tissue macromolecules or is further rapidly oxidized to 2-nitrofluorene and azofluorene. Furthermore, this reaction probably proceeds through a free radical mechanism similar to that of horseradish peroxidase.

Animals↗

PubMed1986

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase.

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.

Animals↗

PubMed1985

Unique cyanide nitrogen-15 nuclear magnetic resonance chemical shift values for cyano-peroxidase complexes. Relevance to the heme active-site structure and mechanism of peroxide activation.

Cyanide ion has been utilized to probe the heme environment of the ferric states of horseradish peroxidase, lactoperoxidase and chloroperoxidase. The 15N-NMR signal for cyanide bound to these enzymes is located in the downfield region from 578 to 412 ppm (with respect to the nitrate ion reference). The corresponding signal for met-forms of hemoglobin, myoglobin and cytochrome c is much further downfield in the 1047-847 ppm region. The signal position for peroxidases is quite invariant with pH in the physiological ranges. The upfield bias for peroxidase chemical shifts must reflect unique trans iron(III) ligand types and/or proximal-group hydrogen bonding or steric effects. Model compound studies reveal a significant upfield cyanide 15N shift with addition of agents capable of hydrogen-bonding to the coordinated cyanide ion. An even more striking upfield shift of 277 ppm is associated with deprotonation of a trans imidazole residue. The distinctive chemical shifts observed for the cyano ligand in peroxidases support the hypothesis that a distal hydrogen-bonding network and perhaps a polar, basic trans ligand are essential for O-O bond activation by peroxidases.

Animals↗

PubMed1988

Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus.

The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.

Azides↗

PubMed1996

Oscillations in the peroxidase-oxidase reaction: a comparison of different peroxidases.

The nonlinear behavior of the peroxidase-oxidase reaction was studied using structurally different peroxidases. For the first time sustained oscillations with peroxidases other than horseradish peroxidase in a single-enzyme system were observed. All peroxidases that showed significant oxidase activity were able to generate sustained oscillations. When adjusting the overall reaction rate, either of the two modifiers 2,4-dichlorophenol or Methylene blue could be omitted from the reaction. Due to the observation of different enzyme intermediates when using different peroxidases, we conclude that the mechanisms responsible for oscillatory kinetics may vary from one peroxidase to the other.

Animals↗

PubMed1985

Spin trapping of the azidyl radical in azide/catalase/H2O2 and various azide/peroxidase/H2O2 peroxidizing systems.

The azidyl radical is formed during the oxidation of sodium azide by the catalase/hydrogen peroxide system, as detected by the ESR spin-trapping technique. The oxidation of azide by horseradish peroxidase, chloroperoxidase, lactoperoxidase, and myeloperoxidase also forms azidyl radical. It is suggested that the evolution of nitrogen gas and nitrogen oxides reported in the azide/catalase/hydrogen peroxide system results from reactions of the azidyl radical. The azide/horseradish peroxidase/hydrogen peroxide system consumes oxygen, and this oxygen uptake is inhibited by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, presumably due to the competition with oxygen for the azidyl radical. Although azide is used routinely as an inhibitor of myeloperoxidase and catalase, some consideration should be given to the biochemical consequences of the formation of the highly reactive azidyl radical by the peroxidase activity of these enzymes.

Azides↗

PubMed1988

Oxygen-17 nuclear magnetic resonance spectroscopic studies of carbonmonoxyperoxidases.

We have obtained oxygen-17 (17O) nuclear magnetic resonance (NMR) spectra of C17O ligands bound to ferrous horseradish peroxidase isozyme A, isozyme C, and ferrous chloroperoxidase, as a function of pH. Our results show that the peroxidases exist in two distinct states, the acidic and alkaline forms, which undergo reversible acid-base-induced transitions characterized by a single pK value. The two forms are characterized spectroscopically in much the same way in all three proteins, suggesting a similar structural origin for the transition process. In particular, the 17O NMR signal of the acidic form is approximately 7 ppm more shielded than that of the alkaline form, and the CO ligand in the acidic form appears to have a smaller 17O nuclear quadrupole coupling constant than that of the alkaline form. We have also obtained the pK values and exchange rates for all three peroxidases. The results indicate that a similar structural change may be involved in the transition process in all three peroxidases.

Carbon Monoxide↗

PubMed1985

Comparison of heme environments and proximal ligands in peroxidases and other hemoproteins through carbon-13 nuclear magnetic resonance spectroscopy of carbon monoxide complexes.

Carbon-13 nuclear magnetic resonance signals for the carbon monoxide ligand in ferrous complexes of horseradish peroxidase, lactoperoxidase, and chloroperoxidase are located respectively at 209.1, 208.3, and 200.8 parts per million from the tetramethylsilane reference. On the basis of previous hemoprotein and model compound studies these resonance positions are consistent with coordination of a proximal histidine ligand in horseradish peroxidase and lactoperoxidase, and coordination of a cysteinyl mercaptide ligand in chloroperoxidase. Carbonyl chemical shift values for acidic and basic horseradish peroxidase isoenzymes are very similar.

Carbon Monoxide↗

PubMed2005

Amphiphilic network as nanoreactor for enzymes in organic solvents.

Enzymes are powerful biocatalysts that work naturally in water but are also active in organic solvents. Here, we present a nanophase-separated amphiphilic network, where an enzyme is entrapped into its hydrophilic domains. A substrate that diffuses into the other, hydrophobic, phase of such a network can access the biocatalyst via the extremely large interface. Entrapped horseradish peroxidase and chloroperoxidase showed dramatically increased activity and operational stability compared to the native enzymes.

Catalysis↗

PubMedDate not supplied

Peroxygenation mechanism for chloroperoxidase-catalyzed N-oxidation of arylamines.

The metabolism of three arylamine substrates by H2O2 in the presence of each of the peroxidative enzymes chloroperoxidase (CPX) and pea seed peroxygenase (PSM) was conducted with normal H2O2 and with 18O-labeled H2O2. The resulting C-nitroso aromatic metabolites were examined by GC-MS methods to determine the extent of 18O incorporation. The arylamine substrates were p-toluidine, 4-chloroaniline, and 3,4-dichloroaniline. For both enzymes, all three arylamines were found to give quantitative incorporation of 18O into their nitroso metabolites when [18O]H2O2 was the oxidant substrate. The introduction of the oxygen atom into 4-chloronitrosobenzene was found to occur during the first step of this process, since it was found that when (4-chlorophenyl)hydroxylamine was employed as the substrate, no significant incorporation of 18O occurred. These observations prove that CPX and PSM cause N-oxidation of primary arylamines via an oxygen transfer from the compound I activated forms of their heme functional groups. Therefore, these peroxidases are correctly called peroxygenases when acting in such a manner. A discussion of the reaction mechanisms for peroxidases and their relation to cytochrome P-450 oxidations is presented.

Amines↗

PubMed2001

Eosinophil markers in blood, serum, and urine for monitoring the clinical course in childhood asthma: impact of budesonide treatment and withdrawal.

BACKGROUND: Markers of airway inflammation are needed for prediction of asthma deterioration and evaluation of disease severity. Few studies have focused on the dynamics of airway inflammation as reflected by the activity of the eosinophils and their proteins after withdrawal of inhaled corticosteroids. OBJECTIVE: Our goal was to investigate the effect of withdrawal of inhaled budesonide on eosinophil count in blood and eosinophil proteins in serum and urine and to relate the levels of these markers to the risk of symptoms of asthma, increased bronchial hyperresponsiveness, and deterioration of lung function. METHODS: Thirty-three children were randomly selected to continue or discontinue use of inhaled budesonide in a double-blind, placebo-controlled study. They were followed up for 4 months with regular analysis of blood, serum, and urine samples; lung function; and methacholine challenges. Eosinophil activity markers were analyzed. Age-matched healthy children provided reference data for all parameters measured. RESULTS: The eosinophil number in blood and eosinophil protein levels in serum (serum eosinophil cationic protein [ECP] and serum eosinophil peroxidase [EPO]) increased significantly in the withdrawal group, and the difference between the groups was significant (P =.02 for all). Twenty-nine percent of the children in the withdrawal group remained symptom free. This subgroup had eosinophil counts at baseline below 350/microL, a serum ECP level below 15 microg/L, and a serum EPO level below 25 microg/L, each of which was related to a low risk of exacerbation (relative risk = 0.37, 0.48, and 0.37 respectively; P <.05 for all). All eosinophil markers were lower in the healthy children than in the symptom-free children with asthma. CONCLUSION: Our data indicate that eosinophil count and/or ECP and EPO levels can be used to estimate the short-term risk of deterioration and the need for corticosteroid treatment in cases of mild and moderate allergic asthma.

Adolescent↗

PubMed1996

The distal glutamic acid as an acid-base catalyst in the distal site of horseradish peroxidase.

The distal His is an essential amino acid residue as a general acid-base catalyst for peroxidase reaction cycle. However, the x-ray structure of chloroperoxidase revealed that Glu is located near the heme, suggesting that the carboxyl group also assists cleavage of O-O bond in peroxides. In this paper, we examined functional and structural properties of a horseradish peroxidase mutant having Glu instead of the distal His. Although this amino acid replacement depressed reaction rate with H2O2 and oxidation activity for guaiacol, the mutant still exhibited much higher activity than mutants in which the distal His was replaced by hydrophobic amino acid. Kinetic measurements suggest that the proton abstraction is decelerated in the mutant due to large fluctuation of the carboxyl group of the distal Glu. Therefore, we can conclude that Glu can be a potent acid-base catalyst for peroxidase reaction cycle, if the carboxyl group can be fixed at the optimum position.

Amino Acid Sequence↗

PubMed2006

C. fumago chloroperoxidase is also a dehaloperoxidase: oxidative dehalogenation of halophenols.

We have examined the H2O2-dependent oxidative dehalogenation of 2,4,6-trihalophenols and p-halophenols catalyzed by Caldariomyces fumago chloroperoxidase (CCPO). CCPO is significantly more robust than other peroxidases and can function under harsher reaction conditions, and so its ability to dehalogenate halophenols could lead to its use as a bioremediation catalyst for aromatic dehalogenation reactions. Optimal catalysis occurred under acidic conditions (100 mM potassium phosphate solution, pH 3.0). UV-visible absorption spectroscopy, high-performance liquid chromatography, and gas chromatography/mass spectrometry clearly identified the oxidized reaction product for CCPO-catalyzed dehalogenation of 2,4,6-trihalophenols as the corresponding 2,6-dihalo-1,4-benzoquinones. This reaction has previously been reported for two His-ligated heme-containing peroxidases (see Osborne, R. L.; Taylor, L. O.; Han, K. P.; Ely, B.; Dawson, J. H. Biochem. Biophys. Res. Commun. 2004, 324, 1194-1198), but this is the first example of a Cys-ligated heme-containing peroxidase functioning as a dehaloperoxidase. The relative catalytic efficiency (turnover number) of CCPO reported herein is comparable to that of horseradish peroxidase (Ferrari, R. P.; Laurenti, E.; Trotta, F. J. Biol. Inorg. Chem. 1965, 4, 232-237). The mechanism of dehalogenation has been probed using p-halophenols as substrates. Here the major product is a dimer with 1,4-benzoquinone as the minor product. An electron-transfer mechanism is proposed that accounts for the products formed from both the 2,4,6-trihalo- and p-halophenols. Finally, we note that this is the first case of a peroxidase known primarily for its halogenation ability being shown to also dehalogenate substrates.

Ascomycota↗

PubMed1978

Chloroperoxidase-catalysed oxidation of 4-chloroaniline to 4-chloronitrosobenze.

The incubation of 4-chloroaniline with chloroperoxidase and H2O2 resulted in a rapid formation of 4-chloronitrosobenzene. This enzymic oxidation displayed a pH optimum at 4.4 with a Km of 8.1x10(-4)M and catalytic-centre activity of 312. The initial rate of the reaction was strongly affected by the presence of halide ions. 4-Chlorophenylhydroxylamine was even more rapidly converted into the nitroso compound. A reaction mechanism is proposed on the basis of currently accepted theory for the catalytic action of chloroperoxidae. A noteworthy aspect of this new reaction is the difference in the products previously reported for the action of classical peroxidases on anilines and the single nitroso product resulting from chloroperoxidase oxidation.

Aniline Compounds↗

PubMed1991

Free radical formation in the oxidation of malondialdehyde and acetylacetone by peroxidase enzymes.

Malondialdehyde, a product of lipid peroxidation, and acetylacetone undergo one-electron oxidation by peroxidase enzymes to form free radical metabolites, which were detected with ESR using the spin-trapping technique. The structures of the radical adducts were assigned using isotope substitution. With both malondialdehyde and acetylacetone and the enzymes myeloperoxidase and chloroperoxidase, carbon-centered radicals were detected. With horseradish peroxidase, a carbon-centered radical was initially trapped and then disappeared with the concomitant appearance of an iminoxyl radical.

Chloride Peroxidase↗