Hydrogen-peroxide Oxidoreductase Research Articles

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and −330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals ( g z 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with g z 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric ( g z 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.

Coconut peroxidase isoenzymes: isolation, partial purification and physicochemical properties

Peroxidases (donor: hydrogen peroxide oxidoreductase; EC 1.11.1.7) from normal and mutant (makapuno) coconut endosperms were compared in terms of their specific activities and isoenzyme patterns, physicochemical properties and ability to oxidize IAA. Makapuno peroxidases have significantly higher activity during the early stages of development and significantly lower activity at maturity when compared with the normal. Six anodic isoenzymes were detected at pH 8.9 from both sources by polyacrylamide gel electrophoresis. Both normal and makapuno crude peroxidases have optimum activity at pH 5.5 and are thermostable over a wide range of temperature. The V max was obtained at 1 × 10 −2 M hydrogen peroxide while the apparent K m was at ca 1 × 10 −3 M hydrogen peroxide. In general, no marked difference was noted between the physicochemical properties of peroxidases from both sources. Isolation and partial purification of the isoenzymes were performed by conventional methods including ammonium sulfate fractionation, DEAE Sephadex A-50 ion exchange chromatography and Sephadex G-200 gel filtration. Peroxidase activity was localized in the 50–95% ammonium sulfate precipitate. Gel filtration resolved three protein peaks with peroxidase (Px) activity, the electrophoretic mobilities of which corresponded to Px3, Px4 and Px5. Px5 was purified 17 600-fold and found to be a tetramer with a native MW of 196000 and a subunit MW of 55000. Px3 and Px4 were monomers with MWs of 36300 and 46800, respectively. IAA oxidase activity was detected in the partially purified peroxidases from developing normal endosperms but not from mature endosperms.

Kinetics of yeast cytochrome c peroxidase compound I formation with modified substrates (peroxybenzoic acids)

The kinetics of formation of Compound I of yeast cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) with a series of peroxybenzoic acids were studied. Reactivity is affected not only by protein ionization, as in the reaction with H 2O 2, but also by substrate ionization. The reactivity of negatively charged substrates is markedly lower than that of uncharged species, implying that electrostatic factors profoundly influence substrate binding. The rate constants for neutral peroxybenzoic acids carrying electron-withdrawing substituents increase with increasing peroxy acid p K a. This behaviour suggests that, as previously discussed for reactions of turnip peroxidases, formation of peroxy anion by ionization of substrate within the active site is kinetically important. The results support the mechanism of cytochrome c peroxidase Compound I formation which has been proposed by Poulos and Kraut (J. Biol. Chem. 225 (1980), 8199–8205) on the basis of enzyme structural studies.

Amino acid sequence around the active-site selenocysteine of rat liver glutathione peroxidase

Glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) from rat liver was purified to at least 95% purity. A peptide of 16 amino acids, including the active-site selenocysteine (SeCys) residue, was isolated from a tryptic digest by reverse-phase HPLC and gel filtration. The amino acid sequence of the first 46 residues of glutathione peroxidase was obtained from the overlapping sequences of the tryptic selenopeptide and the intact subunit. The selenocysteine residue was located at residue 41, and the sequence of the tryptic selenopeptide was Val-Leu-Leu-Ile-Glu-Asn-Val-Ala-Ser-Leu-SeCys-Gly-Thr-ThrThr-Arg. The sequence was analyzed by computer for homology with other proteins, but no closely related sequences were found. Glutathione peroxidase is the first selenoprotein for which sequence data have been obtained, and the methods described should be applicable to mapping and to sequence analysis of Se-containing peptides from other selenoproteins.

Selenium and selenoproteins in the rat kidney

Kidney tissue contains a high concentration of selenium that is not accounted for by the known selenoprotein glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9). In order to investigate the nonglutathione peroxidase selenium, rats were isotopically labeled with [ 75Se]selenite over a 10-day period. After this time half of the 75Se in kidney homogenate was found in the particulate subcellular fractions. The kidney lysosomes contained unusually high levels of 75Se, yet they did not contain correspondingly high levels of glutathione peroxidase activity. Two selenoproteins having molecular weights less than 40 000 were resolved by gel filtration from a kidney supernatant fraction. A third selenoprotein exhibited a molecular weight of 75 000. This protein contained one 75 000 molecular-weight subunit, and its selenium was in the amino acid selenocysteine. The 75 000 molecular-weight protein was chromatographically distinct from glutathione peroxidase. In order to determine if these selenoproteins protect against cadmium toxicity, 109CdCl 2 was administered to rats that were isotopically prelabeled with 75Se. At 3, 25 and 72 h after 109Cd administration, no 109Cd was associated with selenium-containing proteins. Two of the nonglutathione peroxidase selenoproteins were apparently unique to the kidney.

Purification of horse eosinophil peroxidase

Eosinophil peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was isolated in a highly purified form (415/280 nm ratio, 1.05) from horse peripheral biood eosinophils. Eosinophil peroxidase was extracted from intact eosinophils (98–100% purity) or isolated eosinophil granules with 0.05 M acetate buffer (pH 4.7)/0.18 M NaCl and purified by chromatography on Sephadex G-200 and carboxymethylcellulose. Final elution was with 0.05 M acetate buffer (pH 4.7)/1 M NaCl. Horse eosinophil peroxidase is a strongly basic protein with bactericidal properties when combined with H 20 2 and iodide, bromide or, to a lesser degree, chloride.

Peroxidase-catalysed oxidation of chlorophyll by hydrogen peroxide

Chlorophyll is effectively bleached by H 2O 2 in the presence of certain phenols and peroxidase (EC 1.11.1.7) extracted from acetone powders of orange flavedo ( Citrus sinensis). Optimal conditions for chlorophyll: hydrogen peroxide oxidoreductase include: pH, 5.9; [H 2O 2] 222 μM; ionic strength 0.11. A phenol is required and resorcinol is the most effective. Catechol and hydroquinone are inhibitory. Chlorophyll a, chlorophyllide a, and chlorophyll b all have similar V max but K m for chlorophyll a is about one-third that of chlorophyll b, while the K m for chlorophyllide a is about one-half that of chlorophyll a. Pheophytin a was much less reactive than chlorophyll a, and Mg 2+ included in the reaction system did not affect rates of pheophytin destruction.

The protoporphyrin-apoperoxidase complex as a horseradish peroxidase analog: A fluorimetric study of the heme pocket

Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0–8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0–1.6. A shift in the apparent p K by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I − and [Fe(CN) 6] −4, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I −-quenching of the porphyrin fluorescence in the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I −. A mechanism for I − oxidation at an acid pH in the presence of peroxidase has been proposed.

Evidence for heme π cation radical species in compound I of horseradish peroxidase and catalase

Magnetic circular dichroism spectra are reported for the compound I species of beef liver catalase (hydrogenperoxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6) and horseradish peroxidase (donor: hydrogenperoxide oxidoreductase, EC 1.11.1.7) and the π cation radical derivatives of porphyrins that have been suggested as models of the electronic configuration of the heme in the compound I species of these enzymes. Comparison of the magnetic circular dichroism spectra of the compound I species with the spectra of [Co(octaethylporphyrin)]2Br and [Co(octaethylporphyrin)]2ClO 4 indicates that in both the intermediate enzyme species the heme has been oxidized to a π cation radical. While there is a clear distinction between the magnetic circular dichroism spectra of the 2A 2u porphyrin, [Co(III)octaethylporphyrin]2ClO 4, and the 2A 1u porphyrin, [Co(III)octaethylporphyrin]2Br, such specific differences are not observed in the spectra of the two enzymes. Analysis of our data suggests that the ground states in the two enzymes are far more similar than the ground states in the two model porphyrins.

Explanation of anomalous binding kinetics with a high yield immobilized enzyme system

The activities of glucose oxidase (β- d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) from commercial preparations do not give typical adsorption curves upon immobilization on non-porous polyethylenimine-coated glass microbeads. The cause of this effect with glucose oxidase was investigated. Protein binding exhibited a rectangular hyperbolic adsorption isotherm, approaching saturation at high concentrations, however, enzyme activities did not. The isotherm for activities exhibited a maxima which corresponded to less than 50% saturation with regard to total protein adsorption. The enzyme preparation was found to contain small quantities of several low molecular weight impurities as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These impurities apparently compete with glucose oxidase for binding. When large excesses of protein are added to beads, the binding of impurities becomes significant and the amount of enzyme activity per unit of bead is reduced.

The Raman spectra of selenomethionine and selenocystine

The trace element selenium is known to be a part of the enzyme glutathione peroxidase (glutathione-hydrogen peroxide oxidoreductase, E.C. 1.11.1.9). Studies have shown that selenium in the enzyme exists in at least two forms or oxidation states. It is probable that selenium has been incorporated into the enzyme as the selenocysteine amino acid. In the present study, the Raman spectra of selenocystine and selenomethionine have been obtained, structural assignments have been verified, and the behavior of the two selenoamino acids have been monitored under varying conditions of oxidative stress. The assignments will assist in the interpretation of thespectrum of the actual enzyme.

Inactivation of galactosyltransferase by lactoperoxidase and [formula omitted

Galactosyltransferase (UDPgalactose: d-glucose 4-β- d-galactosyltransferase, EC 2.4.1.22) was totally inactivated by iodination with lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). Substrates protected against inactivation. The presence of 10 mM Mn 2+ and 1 mM UDPgalactose gave partial protection which was enhanced by the addition of 10 mM N- acetyl-glucosamine , but not by glucose. These results are consistent with a conformational change upon binding of UDPgalactose. Only monoiodotyrosine and diiodotryosine were identified in the pronase digest of iodinated galactosyltransferase. Galactosyltransferase was also inactivated with N- acetylimidazole and partial activity was restored by treating acetylated galactosyltransferase with hydroxylamine. These results suggest that tyrosine(s) is essential for galactosyltransferase activity.

Oxidized forms of ovine erythrocyte glutathione peroxidase cyanide inhibition of a 4-glutathione:4-selenoenzyme

[ 75Se]Glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) containing 4 mol selenium per mol was isolated in 33% yield using 10% ethanol to stabilize the purified enzyme. When reduced with GSH and rapidly separated from GSH by gel filtration chromatography, GSH peroxidase was eluted in a labile oxidized (iodiacetate-insensitive) form which was stable at 4°C but unstable at 25°C (form A). When GSH-reduced enzyme was allowed to oxidize in the course of dialysis a more stable oxidized form was obtained to oxidize in the course of dialysis a more stable oxidized form was obtained (form C) which was rapidly inactivated by cyanide. Using [ 35S]GSH, form C was shown to contain tightly bound glutathione in approx. equimolar ratio with selenium. The cyanide sensitivity of GSH peroxidase is therefore correlated with the presence of a glutathione moiety in the enzyme. The isolation of GSH peroxidase containing bound glutathione suggests that intermediates containing glutathione bound to selenium may be formed during the catalytic cycle.

The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions

The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyses the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H 2O 2, whereas at higher pH, H 2O 2 inhibits the chlorination reaction. The dissociation constant ( K d ) of the spectroscopically detectable complex and the K m for chloride are considerably smaller than the inhibition constant ( K i ) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the K m for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/H 2O 2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.

Studies on peroxidase-catalysed formation of thyroid hormones on a protein isolated from submaxillary gland

A protein has been solubilized and purified to homogeneity from the microsomal fraction of goat submaxillary gland. This protein can preferentially be iodinated to form triidothyronine and thyroxine with the help of submaxillary peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) solubilized and purified from the same microsomal fraction. The protein can also be isolated from soluble supernatant and was found to be identical to the microsomal protein as judged by their molecular properties as well as the formation of triiodothyronine and thyroxine. The protein has the molecular weight of 120 000 and contains two unequal subunits of molecular weight of 80 000 and 44 000. The molecular weight of the peroxidase is 72 000 and consists of a single polypeptide chain. The enzyme has the R z value of 0.4 and is inhibited by azide and cyanide. Mersalyl, a mercurial, strongly inhibits the enzyme activity while N- ethylmaleimide cannot. The enzyme can catalyze the formation of 62 μmol of I − 3/ min per mg of protein at its optimum pH of 5.2. The apparent K m for H 2O 2 and KI is 0.16·10 −3 M and 1 · 10 −3 M, respectively.

The formation of hydrogen cyanide from histidine in the presence of amino acid oxidase and peroxidase

Conditions were sought to increase the yield of HCN from l-histidine incubated with l-amino acid oxidase ( l-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) from snake venom, and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). Small amounts of histidine and high buffer concentrations favored high HCN yields, which reached a maximum of 72%. Imidazole 4-aldehyde and imidazole 4-carboxylic acid were identified among the reaction products, together with CO 2, NH 3, H 2O 2 and imidazole acetic acid. The CO 2 formed was equal to the histidine oxidized, and to the sum of NH 3 plus HCN formed. The production of HCN was associated with an increased O 2 uptake, which was established from the beginning of the reaction, with no apparent lag and ranged from 1.2 to 1.6 μmol extra O 2 taken up/μmol HCN formed. The system was inhibited by catalase, but added superoxide dismutase caused a small stimulation of both HCN production and O 2 consumption, and a larger stimulation of H 2O 2 accumulation. Added hydroxylamine was cooxidized to nitrite in an amount equimolar with the HCN formed. This nitrite formation was inhibited by superoxide dismutase. The facts could be interpreted in terms of superoxide anion formation during the HCN-producing reaction.

Chemical modification of the ε-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation ( k in ) for the native and modified peroxidase at 56–80°C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, ΔH ≠ and ΔS ≠ , have been also determined. A relationship between the number of modified ε-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.

The interrelationship of superoxide dismutase and peroxidatic enzymes in the red cell

Activities of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) were determined during the course of incubation of red cell suspensions with 1,4-naphthoquinone-2-sulfonic acid. In the absence of glucose, incubation with napthoquinone sulfonate resulted in an inhibition of catalase and superoxide dismutase. The catalase inhibitor, 3-amino-1,2,4-triazole enhanced inactivation of catalase in the presence of naphthoquinone sulfonate and this in turn led to augmented inhibition of superoxide dismutase. The presence of glucose in the incubation medium prevented naphtoquinone sulfonate-induced enzyme inhibition in the absence of aminotriazole, but had little effect in the presence of aminotriazole. The relevance of these findings to the cellular interrelationship of peroxidatic enzymes and superoxide dismutase is discussed.

Modification of the blood-brain barrier: increased concentration and fate of enzymes entering the brain.

The blood-brain barrier of rats was opened reversibly by infusing a hyperosmotic solution of arabinose into the external carotid artery. Permeability was increased maximally in the first 15 min and remained slightly elevated at 1 hr. Osmotic barrier opening significantly increased brain uptake of intravenously injected alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.2.4) (derived from human placenta) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). By injection of 4 X 10(5) units of alpha-mannosidase into an animal, brain activity rose to about twice the normal control activity of the enzyme. After 30 min, activity of administered enzyme in the extracellular space of the brain was calculated to be 30% of the serum concentration. Biochemical and histological studies with horseradish peroxidase showed that exogenously administered enzyme entered the brain extracellular space immediately after barrier opening and was incorporated within neuronal lysosomal packets during the next 24 hr. Measurable peroxidase activity was found in brain as much as 72 hr after osmotic treatment. The results demonstrate that the blood-brain barrier can be reversibly opened to enzymes, that a glycoprotein enzyme is incorporated into neuronal lysosomes, and that the brain may now be considered a potential target for enzyme replacement therapy in heritable metabolic disorders.

Biogenesis of peroxisomes: intracellular site of synthesis of catalase and uricase.

The intracellular site of synthesis of two peroxisomal enzymes of rat liver, uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) and catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6), has been localized on free ribosomes and not membrane-bound ribosomes. Free polysomes and membrane-bound polysomes, prepared by classical cell fractionation techniques from rat liver, were incubated for protein synthesis in a cell-free system derived from rabbit reticulocytes. Characterization of the total translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, as well as by immunoprecipitation with anti-rat albumin anti-serum, confirmed that good separation of the two polysome classes was achieved. Uricase and catalase were immunoprecipitable from translation products directed by free polysomes or phenol-extracted free polysomal mRNA but not from products of membrane-bound polysomes. Furthermore, unlike albumin, nascent uricase and catalase were not cotranslationally segregated by dog pancreas microsomal membranes. The results indicate that uricase and catalase are transferred to the interior of peroxisomes by a post-translational mechanism; an hypothesis is formulated here for the biogenesis of peroxisomes.