Hydrogen-peroxide Oxidoreductase Research Articles

Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules

The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (Glycine max). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H(2)O(2) in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.

Cytochrome c′-type cytochrome- c peroxidase derived from Nitrosomonas europaea

From Nitrosomonas europaea, a cytochrome c′-type haem protein has been purified which has cytochrome- c peroxidase (ferrocytochrome- c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) activity. The protein has haem c and shows the high-spin-type absorption spectra: it shows absorption peaks at 400, 500 and 640 nm in the oxidized form and peaks at 433 and 553 nm in the reduced form. Its spectra are changed into the haemochrome-type at pH higher than 11. The absorption spectra of the CO and NO complexes of its reduced form and the NO complex of its oxidized form resemble the respective spectra of cytochromes c′ from other bacteria. However, the haem protein has some characteristics which distinguish it from other cytochromes c′; (1) the protein has a cytochrome- c peroxidase activity, (2) its Soret band is composed of a single peak, (3) its oxidized form reacts with cyanide and azide easily, and (4) its molecule is composed of a haem-containing protein subunit (15 kDa) and a non-haem protein subunit (10 kDa). The molecular activities of the protein as cytochrome- c peroxidase are 1010 and 243 per min with horse and N. europaea cytochromes c, respectively.

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.

Uterine changes on removal of submaxillary glands in rats (histological changes/uterine peroxidase/blood estradiol level)

Surgical removal of submaxillary gland in immature rats causes a large increase in size and about three to four fold increase in dry and wet weight of uterus compared to that of the sham operated animals of the same age group. Histological examination reveals a significant increase in the diameter of the uterus with considerable elongation of the luminer epithelium from cubical to columnar in the experimental group. Biochemical studies show that the uterine peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), a marker enzyme for uterine growth, increases by ten to fifteen fold on submaxillariectomy and returns almost to the normal level on administration of submaxillary gland extract (105,000 × g supernatant) to the submaxillariectomized animals. Estrogen estimation by radioimmunoassay shows a similar increase of three to four fold on removal of submaxillary glands and decrease almost to the normal value on administration of the submaxillary extract.

Lignocellulose biotransformation with immobilized cellulase, d-glucose oxidase and fungal peroxidases

Three enzymes, cellulase [see 1,4-(1,3;1,4)-β- d-glucan 4-glucanohydrolase, EC 3.2.1.4], d-glucose oxidase (β- d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) immobilized on glass beads, have been incubated with lignocellulose. Fungal peroxidases from Trametes versicolor and Inonotus radiatus when mixed with cellulase and d-glucose oxidase were able to liberate phenolic compounds and d-glucose from lignocellulose. Three lignin monomers were identified. When the immobilized enzymes were incubated individually with lignocellulose they did not degrade lignin .

Thermal denaturation of cytochrome c peroxidase: pH dependence

Upon heating cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) at pH 4 and 5, the enzyme precipitates at 41°C and 51°C, respectively. Incubating the enzyme at lower temperatures causes a slow dissociation of the heme from the protein. The heme precipitates, while the apoprotein remains soluble. Between pH 6 and 8, the native enzyme is converted to a low-spin ferric form upon heating. The Soret maximum shifts from 408 to 414 nm. The midpoint of this transition is pH-dependent, with a value of 46°C at pH 6 decreasing to 29°C at pH 8. At high temperatures the 414 nm form is converted to a species which has a ‘free heme’ spectrum with low absorptivity and Soret maximum at 390 nm. The midpoint temperature of this latter transition is 62°C and 57°C at pH 7 and 8, respectively.

Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent

Soret excitation resonance Raman spectroscopy has been used to characterize dimeric human leukocyte myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) and monomeric bovine spleen green haemoprotein. The spectra of the two proteins, under the same conditions of iron valence and ligation, are essentially identical. Owing to strong symmetry reduction effects, the spectra are more complex than usually observed for haemoproteins. It is possible, however, to assign the high-frequency vibrations and, from these assignments, to determine structural features of the iron chromophores. In the resting protein, the iron adopts a six-coordinate high-spin configuration in both proteins; cyanide addition produces six-coordinate low-spin species, and in the ferrous enzymes the iron appears to be five-coordinate and high-spin. The proteins are stable to laser excitation and do not photoreduce under illumination. No evidence is found for unusual peripheral substituents, such as formyl or protonated Schiff's base group, in conjugation with the main chromophore in the native protein. The vibrational data are consistent with an iron chlorin chromophore, although other electronic effects, in addition to those produced by porphyrin ring reduction, are necessary to account for the optical properties of the proteins. The similarity in Raman spectra for myeloperoxidase and green haemoprotein indicates that the two iron sites in myeloperoxidase are equivalent.

The nitrosyl compounds of ferrous animal haloperoxidases

Human myeloperoxidase, human eosinophil peroxidase and bovine lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) reduced with ascorbic acid form nitrosyl compounds which show rhombic EPR signals centered at g = 2. Using 14NO ( I N = 1), the central resonance signal exhibited a hyperfine structure of nine lines originating from a triplet with a small hyperfine splitting ( A II( z) = 0.69 mT for myeloperoxidase and 0.73 mT for eosinophil peroxidase and lactoperoxidase) superimposed upon a triplet with a larger hyperfine splitting ( A I ( z) = 2.34, 2.32 and 2.09 mT for myeloperoxidase, eosinophil peroxidase and lactoperoxidase, respectively). Using 15NO ( I N = 1 2 ), the nitrosyl compound of ferrous myeloperoxidase and ferrous lactoperoxidase showed a doublet of triplets superimposed upon the central resonance signal. These results demonstrate that a nitrogen nucleus is present at the fifth ligand position of the haem iron in these peroxidases.

Caractéristiques fonctionnelles des activités peroxydasiques des feuilles et cals d'une plante à métabolisme acide crassulacéen, Pedilanthus tithymaloides variegatus, Euphorbiaceae

Peroxidases (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7) from leaf tissues and calli were extracted and compared in terms of their specific activities at different pH and temperatures, their isoenzyme patterns and physicochemical properties. Three groups of enzyme activities were detected and their purification was performed by conventional methods (ammonium sulfate fractionation, ion-exchange chromatography, gel filtration, affinity chromatography).

Rainbow trout liver microsomal lipid peroxidation the effect of purified glutathione peroxidase, glutathione S-transferase and other factors

Glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9) was purified approximately 600-fold from rainbow trout liver soluble fraction and its activity in the NADPH microsomal lipid peroxidation system tested. The enzyme has an approximate molecular weight of 100000, contains four subunits and four atoms of selenium per mol protein. No selenium-independent glutathione peroxidase activity could be attributed to glutathione S-transferase (EC 2.5.1.18) in trout liver. Glutathione peroxidase together with glutathione (GSH) did not provide any additional protection in the in vitro liver microsomal lipid peroxidation system over and above that provided by GSH alone. Microsomal lipid peroxidation was, however, reduced by a partially purified glutathione S-transferase together with GSH. The protection provided by dialysed liver cytosol in this system was not GSH-dependent, showing that other factors in addition to glutathione S-transferase are involved. Of other possible factors, vitamin E reduced lipid peroxidation in this system. Concentrations of vitamin E in microsomes before and after peroxidation in vitro indicated that protective cytosolic factor(s) act prior to the termination of the free radical chain reactions effected by vitamin E. A GSH-dependent protective factor was present in microsomal protein, malondialdehyde formation in the in vitro microsomal system being markedly reduced in the presence of 5 mM GSH but not significantly lowered by 1 mM GSH.

A kinetic study of the reaction between human myeloperoxidase, hydroperoxides and cyanide inhibition by chloride and thiocyanate

The reaction between myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), hydrogen peroxide and ethyl hydroperoxide was investigated using the stopped-flow technique. Like other peroxidases, myeloperoxidase forms two sequential peroxide compounds. The pH-dependence of the apparent second-order rate constant of compound I formation shows that there is an acid/base group on the enzyme with a p K a of 4.30 ± 0.15, which — when protonated — prevents the reaction of the enzyme with peroxides. The rate constants for the formation of compound I by hydrogen peroxide and ethyl hydroperoxide are (2.3 ± 0.1)·10 7 M −1·s −1 and (2.8 ± 0.3) · 10 5 M −·s −1, respectively. The binding of cyanide to myeloperoxidase ( k 1 = (1.30 ± 0.05)·10 6 M −1·s −1) is also regulated by an acid/base group with a p K a of 4.00 ± 0.05 as is the case with hydrogen peroxide; also, only the protonated uncharged form of cyanide reacts with the enzyme. From their effects on the binding of cyanide to the enzyme it is concluded that chloride and thiocyanate bind to myeloperoxidase only when the acid/base group is protonated. The pH-dependence of the dissociation constant of the myeloperoxidase-chloride complex obtained from the spectral changes induced by chloride is the same as observed in the inhibition by chloride of the binding of cyanide. It is concluded that hydrogen peroxide, cyanide, chloride and thiocyanate bind at the same site on the enzyme.

Use of a bioelectrochemical cell for the synthesis of (bio)chemicals

A bioelectrochemical cell containing either d-glucose oxidase (β- d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) or xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) plus dichlorophenol-indophenol as electron acceptor in one half-cell, and chloroperoxidase (chloride:hydrogen-peroxide oxidoreductase, EC 1.11.1.10) in the other half-cell is described. Due to a combination of chemical, biochemical and electrochemical reactions, electricity and specific (bio)chemicals can be produced in the cell simultaneously and in both compartments. Furthermore, the oxidases in a bioelectrochemical cell are not inactivated by H 2O 2 and as a result the operational lifetimes of the oxidases were increased about five-fold.

Favism: a hemolytic disease associated with increased superoxide dismutase and decreased glutathione peroxidase activities in red blood cells.

Red blood cells of favism patients with acute hemolytic crisis have markedly more superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and less glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) than either normal controls, glucose-6-phosphate dehydrogenase-deficient subjects or favism patients outside hemolytic crisis. This altered value of the two enzyme activities is not due to increased reticulocyte content of blood. The electrophoretic triplet pattern of superoxide dismutase is also changed, with significant increase of the most positively charged band. Similar modifications of the two enzyme activities are observed after treatment of normal red blood cells with high concentrations of divicine and ascorbate, which are redox compounds that are contained in fava seeds. This treatment produces no hemolysis, but leads to hemolysis if the treated cells are resuspended in the homologous plasma. These results suggest a possible role of active oxygen species in the development of favism.

The nitrosyl compound of ferrous lactoperoxidase

The nitrosyl compound of reduced bovine lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) has been prepared using two methods of initial reduction prior to exposure to NO; namely, dithionite and electrochemical reduction. In both cases, a rhombic EPR spectrum with a three-line hyperfine splitting of the central band was obtained at 80 K. This indicates an interaction between the unpaired electron and single 14N nucleus of the NO radical. It is concluded that the iron-histidine bond, shown by magnetic circular dichroism (Sievers, G., Gadsby, P.M.A., Peterson, J. and Thomson, A.J. (1983) Biochim. Biophys. Acta 742, 659–668) to be present in the oxidised state of the enzyme, is broken in the ferrous nitrosyl derivative.

Vitamin C. Stimulates the chlorinating activity of human myeloperoxidase

Ascorbic acid (vitamin C) was found to stimulate the chlorinating activity of human myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) 3-fold in vitro and to shift the pH optimum of the reaction to higher pH values. These effects are due to the conversion by ascorbic acid of inactive compound II formed during turnover into native enzyme.

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase

The anion-binding characteristics of resting and half-reduced Pseudonomas cytochrome c perosidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (Rönnberg M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197–204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the g z values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 μM, 2.5 mM and 0.13 M, respectivelty. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.

Some properties of human eosinophil peroxidase, a comparison with other peroxidases

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71 000 ± 1000. The enzyme is composed of two subunits, of M r 58 000 and 14 000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for ϵ 412nm of 110 ± 4 m −1 · cm −. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry ( g x = 6.50; g y = 5.40; g z = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.

Steady-state kinetics of yeast cytochrome c peroxidase catalyzed oxidation of inorganic reductants by hydrogen peroxide

Rates of yeast cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) catalyzed oxidation of bis(tripyridine)cobalt(II)ion, penta(amine)pyridineruthenium(II) ion and ferrocyanide ion by hydrogen peroxide have been found to obey the empirical equation: Initial rate= k 0[S]+ k 1k 2[S] k 1[S]+k 2 [CcP] in the pH range 5 to 8, and at saturating H 2O 2 concentrations. ([S] and [CcP] are the concentrations of the reductant and the enzyme, respectively.) Values of k 2 were found to be independent of the reductant. The term k 0[ S] is only significant with the cobalt and ruthenium complexes at high pH. The mechanism proposed to account for this rate equation differs significantly from previous mechanistic proposals. In particular, the rate data require the assignment of the rate-limiting step at high substrate concentrations to a slow electron-transfer within the enzyme, and not, as previously suggested, to saturation of substrate binding to the enzyme. Also, the term K 0[ S] implies that the reative substrates, including the natural substrate (yeast cytochrome c), react with the hydrogen peroxide-heme complex and not with the radical species formed by reaction with hydrogen peroxide in the absence of reductants.

In vitro synthesis of glutathione peroxidase from selenite Translational incorporation of selenocysteine

The synthesis of glutathione peroxidase from [ 75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [ 75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [ 3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [ 75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [ 75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase

Modifiers of free radicals inhibit in vitro the oncogenic actions of x-rays, bleomycin, and the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

Using short-term cultures of hamster embryo cells, we have examined the effects of the free-radical scavenger superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and the enzyme catalase (hydrogen-peroxide:hydrogenperoxide oxidoreductase, EC 1.11.1.6) on x-ray- and bleomycin-induced transformation and on the enhancement of radiogenic transformation by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). We find that superoxide dismutase inhibits (i) transformation induced by x-ray and bleomycin and (ii) promotional action of TPA in vitro. The results suggest that the oncogenic action of x-rays and bleomycin and the enhancement of oncogenic transformation by TPA are mediated in part by free radicals. The findings also suggest that superoxide dismutase can serve as an inhibitor of oncogenesis and that its actions, as seen in this in vitro system, are most predominantly on inhibiting late events in the progression of cellular transformation--those associated with promotion.