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PubMed2006

Structures of the high-valent metal-ion haem-oxygen intermediates in peroxidases, oxygenases and catalases.

Peroxidases, oxygenases and catalases have similar high-valent metal-ion intermediates in their respective reaction cycles. In this review, haem-based examples will be discussed. The intermediates of the haem-containing enzymes have been extensively studied for many years by different spectroscopic methods like UV-Vis, EPR (electron paramagnetic resonance), resonance Raman, Mössbauer and MCD (magnetic circular dichroism). The first crystal structure of one of these high-valent intermediates was on cytochrome c peroxidase in 1987. Since then, structures have appeared for catalases in 1996, 2002, 2003, putatively for cytochrome P450 in 2000, for myoglobin in 2002, for horseradish peroxidase in 2002 and for cytochrome c peroxidase again in 1994 and 2003. This review will focus on the most recent structural investigations for the different intermediates of these proteins. The structures of these intermediates will also be viewed in light of quantum mechanical (QM) calculations on haem models. In particular quantum refinement, which is a combination of QM calculations and crystallography, will be discussed. Only small structural changes accompany the generation of these intermediates. The crystal structures show that the compound I state, with a so called pi-cation radical on the haem group, has a relatively short iron-oxygen bond (1.67-1.76A) in agreement with a double-bond character, while the compound II state or the compound I state with a radical on an amino acid residue have a relatively long iron-oxygen bond (1.86-1.92A) in agreement with a single-bond character where the oxygen-atom is protonated.

Catalase↗
PubMed2006

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants.

In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.

Amino Acid Substitution↗
PubMed2004

The 2.0 A resolution crystal structure of prostaglandin H2 synthase-1: structural insights into an unusual peroxidase.

Prostaglandin H2 synthase (EC 1.14.99.1) is an integral membrane enzyme containing a cyclooxygenase site, which is the target for the non-steroidal anti-inflammatory drugs, and a spatially distinct peroxidase site. Previous crystallographic studies of this clinically important drug target have been hindered by low resolution. We present here the 2.0 A resolution X-ray crystal structure of ovine prostaglandin H2 synthase-1 in complex with alpha-methyl-4-biphenylacetic acid, a defluorinated analog of the non-steroidal anti-inflammatory drug flurbiprofen. Detergent molecules are seen to bind to the protein's membrane-binding domain, and their positions suggest the depth to which this domain is likely to penetrate into the lipid bilayer. The relation of the enzyme's proximal heme ligand His388 to the heme iron is atypical for a peroxidase; the iron-histidine bond is unusually long and a substantial tilt angle is observed between the heme and imidazole planes. A molecule of glycerol, used as a cryoprotectant during diffraction experiments, is seen to bind in the peroxidase site, offering the first view of any ligand in this active site. Insights gained from glycerol binding may prove useful in the design of a peroxidase-specific ligand.

Animals↗
PubMed2004

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves.

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.

Amino Acid Sequence↗
PubMed2004

Expression analysis of the Arabidopsis peroxidase multigenic family.

Class III peroxidases form a numerous multigenic family in higher plants, whose expression is particularly sensitive to internal or external events. Arabidopsis thaliana genome harbours 73 genes encoding peroxidases. Since they exhibit homologies ranging from 28% to 93% at the nucleotide level, the risk of cross-hybridisation may be important when measuring the level of transcripts by blotting techniques, using whole cDNA sequences. We developed a procedure to assess the expression of all peroxidase genes on one membrane, with a high specificity. The method was based on the determination for each gene of a short specific sequence (amplicon) exhibiting at the most 70% homology with any other sequences of the Arabidopsis genome. Amplicons specific for each of the 73 peroxidase genes and two pseudogenes were blotted on a nylon membrane that was hybridised with radiolabelled cDNA libraries prepared from mRNAs of Arabidopsis roots, stems, leaves and flowers. Many genes were expressed at a low level, often in all organs, while sixteen genes were rather strongly expressed, in two to four organs. Some genes with no ESTs reported in databases were found to be expressed and this was confirmed by RT-PCR. Isoelectric focusing analysis revealed that the isoperoxidase pattern was similar in leaves, stems and flowers, but was quite different in roots. To our knowledge, only one similar study has been performed on the cytochrome P450 family, using microarrays, but this is the first work describing the expression profile of a whole large multigenic family using specific macroarrays.

Arabidopsis↗
PubMed2003

Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies.

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., "hot" peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 degrees C, pH 7.0). We determined rate constants approaching 2 x 10(7) and 5 x 10(5)M(-1)s(-1) for compound I and compound II reduction, respectively. We also determined k(app) values for steady-state capsaicinoid oxidation approaching 8 x 10(5)M(-1)s(-1) (25 degrees C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.

Animals↗
PubMed2003

Determination of salivary peroxidase activity in human mixed whole saliva.

Mixed whole saliva contains salivary peroxidase (SPX) and myeloperoxidase (MPO), thus it is important to discriminate between the two peroxidases in order to understand their functions in the oral cavity. We developed a method to measure SPX activity in mixed whole saliva using an oxygen electrode. According to our results, when 50% of the peroxidase activity in saliva was due to MPO, determined using a typical substrate for peroxidase guaiacol, almost all oxygen evolved was due to SPX. We propose that measurement of H(2)O(2)-dependent oxygen evolution is a useful method for determining SPX activity in whole saliva.

Electrochemistry↗
PubMed2003

Human salivary peroxidase-catalyzed oxidation of nitrite and nitration of salivary components 4-hydroxyphenylacetic acid and proteins.

Human saliva contains high activities of peroxidase and high concentrations of nitrite (about 0.2 mM in average). If H2O2 is provided by bacteria and leukocytes in the oral cavity, peroxidase-dependent formation of reactive nitrogen species, which can nitrate phenolics like 4-hydroxyphenylacetic acid (HPA) and tyrosine residues in salivary proteins, is possible. H2O2-dependent oxidation of nitrite and H2O2-dependent nitration of HPA were observed in dialyzed saliva and by partially purified salivary peroxidase (SPX). The nitration was inhibited by a physiological electron donor to salivary peroxidase, SCN-. When concentrations of H2O2 and nitrite were increased, nitration of HPA was also observed in control (non-dialyzed) saliva. In addition, H2O2-dependent nitration of tyrosine residues in salivary proteins was observed in dialyzed saliva as an increase in absorbance around 420 nm at pH 7.2. Kinetic studies of the increase in absorbance indicated that sulfhydryl groups in salivary proteins as well as glutathione, ascorbate, urate and SCN- could inhibit the nitration. Since the nitration of proteins can lead to impairment of their functions, it is discussed how the oral cavity is protected from the damages caused by reactive nitrogen species under normal conditions and also discussed that reactive nitrogen species generated by the H2O2/nitrite/peroxidase system can participate in the host defence mechanism in the oral cavity.

Humans↗
PubMed1988

Structure and regulation of a lignin peroxidase gene from Phanerochaete chrysosporium.

We have been investigating the structure and regulation of the lignin peroxidase genes from Phanerochaete chrysosporium. The gene for lignin peroxidase isozyme H8 was isolated and sequenced. The gene is split into nine exons and eight introns. The introns have consensus sequences both at the splice junctions and at an internal element which may be involved in the splicing mechanism. The promoter contains the eukaryotic consensus elements: a TATA box at -78 and a ACAAT box at -106. Transcription initiates downstream from these sequences as verified by S1 nuclease mapping. Induction of the genes for the lignin peroxidase isozymes during growth under nitrogen and under carbon limitation were compared by analyses of RNA and protein. The results indicate that the lignin peroxidase isozymes are differentially regulated in response to environmental stress.

Base Sequence↗
PubMed1988

Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver.

To determine the effect of Se status on the level of mRNA for Se-dependent glutathione peroxidase (EC 1.11.1.9), rats were fed either a Se-deficient torula yeast diet (less than 0.02 mg Se/kg diet) or a Se-adequate diet (+0.2 mg Se/kg as Na2SeO3) for greater than 135 d. Liver glutathione peroxidase activity was 0.025 for Se-deficient versus 0.615 EU/mg protein for Se-adequate rats. Total liver RNA and polyadenylated RNA were isolated and subjected to Northern blot analysis using a 700 bp DNA probe from cloned murine glutathione peroxidase. Autoradiography showed that Se-deficient liver had 7-17% of the mRNA for glutathione peroxidase present in Se-adequate liver, suggesting that Se status may regulate the level of mRNA for this selenoenzyme.

Actins↗
PubMed2001

Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: role of quinone methide in DNA adduct formation.

We have investigated the activation of p-cresol to form DNA adducts using horseradish peroxidase, rat liver microsomes and MnO(2). In vitro activation of p-cresol with horseradish peroxidase produced six DNA adducts with a relative adduct level of 8.03+/-0.43 x 10(-7). The formation of DNA adducts by oxidation of p-cresol with horseradish peroxidase was inhibited 65 and 95% by the addition of either 250 or 500 microM ascorbic acid to the incubation. Activation of p-cresol with phenobarbital-induced rat liver microsomes with NADPH as the cofactor; resulted in the formation of a single DNA adduct with a relative adduct level of 0.28+/-0.08 x 10(-7). Similar incubations of p-cresol with microsomes and cumene hydroperoxide yielded three DNA adducts with a relative adduct level of 0.35+/-0.03 x 10(-7). p-Cresol was oxidized with MnO(2) to a quinone methide. Reaction of p-cresol (QM) with DNA produced five major adducts and a relative adduct level of 20.38+/-1.16 x 10(-7). DNA adducts 1,2 and 3 formed by activation of p-cresol with either horseradish peroxidase or microsomes, are the same as that produced by p-cresol (QM). This observation suggests that p-cresol is activated to a quinone methide intermediate by these activation systems. Incubation of deoxyguanosine-3'-phosphate with p-cresol (QM) resulted in a adduct pattern similar to that observed with DNA; suggesting that guanine is the principal site for modification. Taken together these results demonstrate that oxidation of p-cresol to the quinone methide intermediate results in the formation of DNA adducts. We propose that the DNA adducts formed by p-cresol may be used as molecular biomarkers of occupational exposure to toluene.

Animals↗
PubMed1998

Peroxidase-labelling of human serum transferrin by conjugation to oligosaccharide moieties.

The generation of reactive aldehydes on the carbohydrate moieties of the human serum transferrin was performed by a derivatization procedure based on the mild oxidation with sodium periodate and subsequent reaction with peroxidase hydrazide. The synthesized conjugate was compared to that obtained by modification of the amino acid side chains of transferrin. The conjugate reaction mixture assayed by SDS-PAGE consisted, besides unreacted compounds, of three main bands, corresponding to a molar ratio transferrin:peroxidase of 1:1, 1:2, 1:3. After blotting, these bands were identified by either anti-peroxidase and anti-transferrin antibodies on nitrocellulose membrane. ELISA detection method showed that the conjugate via oligosaccharide moieties (glycans) was still recognized not only by the anti-transferrin antibodies but also by the specific cellular receptor, while the conjugate via amino acids failed to display this latter ability. The different behaviour can be probably due to a significant damage of the protein structure or, possibly, to the peroxidase binding at sites recognized by the receptor. The results reported here indicate that the conjugation procedure through glycans leads to stable and selected transferrin-conjugates fully exhibiting their biological activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1976

Human saliva peroxidase: microanalytical isoelectric fractionation and properties in normal persons and in cases with neuronal ceroid-lipofuscinosis.

Human saliva contains a high peroxidase activity that can be estimated spectrophotometrically with the hydrogen donor p-phenylenediamine and the substrate hydrogen peroxide from 20 mul of material. The pH optimum of the enzyme with citrate-phosphate buffer is 5.5. After microanalytical isoelectric fractionation 3 main isoenzyme components at pI 8.6, 6.5 and 4.3, and a number of isoenzyme subfractions at pI 9.5, 7.3 and 3.8 are detectable. In 3 patients with the juvenile form of neuronal ceroid-lipofuscinosis (type Spielmeyer-Vogt), in which a deficiency of leukocyte peroxidase had been reported by other authors, both the total activity of saliva peroxidase and the activity of individual isoenzymes were found to be within normal limits. These findings are not consistent with a generalized peroxidase deficiency in this disease.

Ceroid↗
PubMed1988

Definitive characterization of uric acid as an interferent in peroxidase indicator reactions and a proposed mechanism of action.

We have characterized and identified uric acid as an interferent to peroxidase catalyzed reactions where hydrogen peroxide is generated at relatively low concentrations. The implications of these findings are important for those utilizing peroxidase as an indicator reaction where low primary substrate concentrations require their preliminary extraction or chemical modification. We have shown that the elimination of uric acid as an interferent from biologic fluid obviates the necessity for such treatment. In amniotic fluid, our data suggests that uric acid represents the only interference to peroxidase-catalyzed reactions, especially when using p-substituted phenols as proton donors. The removal of uric acid has been shown to eliminate hydrogen peroxide reduction and should allow for an increase in sensitivity and specificity for measurements incorporating a peroxidase-coupled indicator reaction, hence, more effective use of these reaction sequences. To our knowledge, this is the first report of a mechanism to eliminate hydrogen peroxide reduction in amniotic fluid.

Amniotic Fluid↗
PubMed2000

Assay of creatinine using the peroxidase activity of copper-creatinine complexes.

OBJECTIVES: It was our goal to develop a urine dipstick that could measure creatinine with a peroxidase reaction. The simultaneous measurement of albumin and creatinine permits the estimation of the 24-h albumin excretion, an important value in judging existing or likely development of renal failure. A highly sensitive dye-binding dipstick method for albumin exists, and a suitable dipstick for the assay for urine creatinine is described here. METHODS: Copper-creatinine and iron-creatinine complexes have peroxidase activity. With 3,3',5,5'-tetramethylbenzidine (TMB), and diisopropyl benzene dihydroperoxide (DBDH); the peroxidase activity of copper-creatinine and iron-creatinine complexes can be demonstrated. This reaction was used in the assay of urine creatinine either in solution or by a suitably impregnated urine dipstick. RESULTS: Our method based on the peroxidase activity of the copper-creatinine complex has an analytical range for creatinine of 100 mg/L (0.884 mmol/L) to 3000 mg/L (26.52 mmol/L). The creatinine assay is free from most interfering compounds that may be present in urine. Hemoglobin is an interferent, and its effects can be reduced but not eliminated by the addition of 4-hydroxy-2-methyl quinoline. We do not recommend using the dipsticks when visible blood is present or if the dipstick blood test is positive. The copper-creatinine complex oxidizes ascorbic acid; however, we were able to modify the reaction conditions so that ascorbic acid at < 4.4 g/L does not interfere. We found good agreement on fresh urines between the creatinine dipstick results and those by a standard rate-Jaffe cuvet method for creatinine. DISCUSSION: With the simultaneous measurement of creatinine and albumin in urine, the albumin/creatinine ratio can be determined effectively reducing or eliminating the occasional false-negative and false-positive result in those with dilute or concentrated urines, respectively. The dipstick test for these analytes permits the simple identification of individuals with possible albuminuria and could serve well in a point-of-care setting.

Amino Acids↗
PubMed2002

H(2)O(2) generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis.

Characterization of lignified Zinnia elegans hypocotyls by both alkaline nitrobenzene oxidation and thioacidolysis reveals that coniferyl alcohol units are mainly found as part of 4-O-linked end groups and aryl-glycerol-beta-aryl ether (beta-O-4) structures. Z. elegans hypocotyls also contain a basic peroxidase (EC 1.11.1.7) capable of oxidizing coniferyl alcohol in the absence of H(2)O(2). Results showed that the oxidase activity of the Z. elegans basic peroxidase is stimulated by superoxide dismutase, and inhibited by catalase and anaerobic conditions. Results also showed that the oxidase activity of this peroxidase is due to an evolutionarily gained optimal adaptation of the enzyme to the microM H(2)O(2) concentrations generated during the auto-oxidation of coniferyl alcohol, the stoichiometry of the chemical reaction (mol coniferyl alcohol auto-oxidized/mol H(2)O(2) formed) being 0.496. These results therefore suggest that the H(2)O(2) generated during the auto-oxidation of coniferyl alcohol is the main factor that drives the unusual oxidase activity of this highly conserved lignin-synthesizing class III peroxidase.

Chromatography, High Pressure Liquid↗
PubMed1997

Proton pumping by cytochrome c oxidase is coupled to peroxidase half of its catalytic cycle.

The four-electron reaction cycle of cytochrome oxidase is comprised of an eu-oxidase phase in which the enzyme receives the first two electrons and reduces oxygen to bound peroxide and a peroxidase phase in which the peroxy state formed in the eu-oxidase half of the cycle is reduced by the 3rd and 4th electrons to the ferryl-oxo state and oxidized form, respectively. Here we show that the ferrocyanide-peroxidase activity of cytochrome c oxidase incorporated in phospholipid vesicles is coupled to proton pumping. The H+/e- ratio for the ferrocyanide-peroxidase partial reaction is twice higher than for the overall ferrocyanide-oxidase activity and is close to 2. These results show that proton pumping by COX is confined to the peroxidase part of the enzyme catalytic cycle (transfer of the 3rd and 4th electron) whereas the eu-oxidase part (transfer of the first two electrons) may not be proton pumping.

Animals↗
PubMed1998

Requirement of intact human ceruloplasmin for the glutathione-linked peroxidase activity.

Structural integrity may be needed for the glutathione-linked peroxidase activity of human ceruloplasmin. Intact human ceruloplasmin has a potent peroxidase property to decompose H2O2 in the presence of reduced glutathione. However, the fragment of approximately 116000 Da produced by proteolytic degradation had less than one-third of the glutathione-linked peroxidase activity of intact ceruloplasmin. When further proteolysis occurred, glutathione-linked peroxidase activity of human ceruloplasmin disappeared. In contrast, ceruloplasmin (116000 Da and <96000 Da) fragmented by proteolysis significantly removed H2O2 irrespective of the presence of reduced glutathione. Although proteolytic fragmentation of ceruloplasmin occurs, the antioxidant activity of ceruloplasmin that prevents DNA strand breaks in a metal-catalyzed reaction system was significantly maintained.

Antioxidants↗