Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Role of peroxidase in lignification of tobacco cells : I. Oxidation of nicotinamide adenine dinucleotide and formation of hydrogen peroxide by cell wall peroxidases

The two peroxidase isoenzyme groups (G_I and G_III) localized in the cell walls of tobacco (Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H₂O₂ formation. Peroxidases of the G_III group are slightly more active than those of the G_I group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H₂O₂ required the presence of Mn²⁺ and a phenol as cofactors. The addition of H₂O₂ to the reaction mixture accelerated subsequent NADH-dependent H₂O₂ formation. In the presence of both cofactors or Mn²⁺ alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H₂O₂ accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H₂O₂ formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88).     

An extracellular NADH-oxidizing peroxidase produced by a lignin-degrading basidiomycete,Phanerochaete chrysosporium

A lignin-degrading basidiomycete, Phanerochaete chrysosporium, produces an extracellular peroxidase which in turn produces H₂O₂ by catalyzing the oxidation of NADH and NADPH. The high enzyme activity was observed in the culture grown under nutrient nitrogen limitation (low-N) and high oxygen tension (high-O₂). The enzyme activity was absent in non-ligninolytic agitated culture and in the cultures of non-ligninolytic mutant strains of this organism. The culture method using polyurethane foam cubes as a support for the growing mycelia showed the beneficial effect of producing a large amount of the enzyme. The enzyme is capable of catalyzing the oxidation of NADH and NADPH in the absence of added H₂O₂, and its activity was inhibited strongly by catalase and superoxide dismutase. It is suggested that this peroxidase participates in the ligninolytic system of Phanerochaete chrysosporium as a donor of H₂O₂, which is required for the lignin-peroxidase reaction, by oxidizing extracellular NADH and NADPH.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Effectiveness of phenoxyl radicals generated by peroxidase/H2O2-catalyzed oxidation of caffeate, ferulate, and p-coumarate in cooxidation of ascorbate and NADH

The rate of ascorbate and nicotinamide adenine dinucleotide plus hydrogen (NADH) cooxidation (i.e., their nonenzymic oxidation by peroxidase/H₂O₂-generated phenoxyl radicals of three hydroxycinnamates: caffeate, ferulate and p-coumarate) was studied in vitro. The reactions initiated by different sources of peroxidase (EC 1.11.1.7) [isolates from soybean (Glycine max L.) seed coat, maize (Zea mays L.) root-cell wall, and commercial horseradish peroxidase] were monitored. Native electrophoresis of samples and specific staining for peroxidase activity revealed various isoforms in each of the three enzyme sources. The peroxidase sources differed both in the rate of H₂O₂-dependent hydroxycinnamate oxidation and in the order of affinity for the phenolic substrates. The three hydroxycinnamates did not differ in their ability to cooxidize ascorbate, whereas NADH cooxidation was affected by substitution of the phenolic ring. Thus, p-coumarate was more efficient than caffeate in NADH cooxidation, with ferulate not being effective at all. Metal ions (Zn²⁺ and Al³⁺) inhibited the reaction of peroxidase with p-coumarate and affected the cooxidation rate of ascorbate and the peroxidase reaction in the same manner with all substrates used. However, inhibition of p-coumarate oxidation by metal ions did not affect NADH cooxidation rate. We propose that both the ascorbate and NADH cooxidation systems can function as mechanisms to scavenge H₂O₂ and regenerate phenolics in different cellular compartments, thus contributing to protection from oxidative damage. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Scopoletin: A substrate for an isoperoxidase from Nicotiana tabacum tissue culture W-38

Scopoletin was found to be a substrate for a single anodic isoperoxidase isolated from tobacco callus tissue W-38. Isolation of this peroxidase was accomplished using DEAE-cellulose chromatography. This isoperoxidase catalysed the destruction of scopoletin in the presence of H₂O₂ only. An enzyme assay for the scopoletin reaction was developed. The pH optimum of the enzyme was 5·5 and the apparent K_ms for scopoletin and H₂O₂ were 0·6 and 0·9 rnM respectively.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

On the role of superoxide in reactions catalyzed by rubredoxin of Pseudomonas oleovorans

In the presence of NADH, and the reductase and rubredoxin components of the ω-hydroxylation system of $\text{Pseudomonas oleovorans}$, epinephrine is oxidized to adrenochrome at pH 7.8, and the reaction is strongly inhibited by the addition of superoxide dismutase (SDM). Boiled SDM has no effect on the reaction rate. The oxidation reaction is oxygen-dependent, and approximately 1 mole of H₂O₂ is produced per mole of O₂ consumed. The stoichiometry between NADH oxidation and adrenochrome formation is approximately 2:1. Epoxidation and epinephrine oxidation are mutually competitive reactions, despite the fact that the epoxidation reaction is not stimulated by a superoxide generating system nor inhibited by SDM.     

Hydroxamate-activated peroxidases in potato tuber callus. Interaction with the determination of the cytochrome and the alternative pathways

In potato (Solatium tuberosum L. cv. Bintje and Doré) callus a very active hydrox-amate-stimulated NADH-dependent O₂-uptake develops during the growth of the callus, which is caused by a peroxidase. More than 95% of the peroxidase activity is found in the 40000 g supernatant. The total activity may be as high as 1000 times the respiratory acitivity of the callus tissue. At least two fractions, obtained by Sephadex gel filtration, can be distinguished showing this peroxidase activity, one of about 15 kDa and one > 50 kDa. The main properties of both fractions are: a) Hydroxamate at 0.2–0.5 mM gives half-maximal stimulation. Maximal stimulation is observed with 1–3 mM benzhydroxamate (BHAM) and 1–15 mM salicylhydroxamate (SHAM). Higher concentrations, especially of BHAM, give less or no stimulation. b) Hydroxamates are not consumed during the reaction. c) Both NADH and NADPH can serve as the electron donor for the reaction. The affinity for NAD(P)H is very low (K_m near 10 mM). In the absence of hydroxamates NAD(P)H is only slowly oxidized, with an even lower affinity. d) The peroxidase can carry out two reactions: an O₂-consuming and a H₂O₂-consuming reaction. In both reactions one NAD(P)H is consumed. In the first reaction H₂O₂ is formed which can be consumed in the second reaction, resulting in an overall stoichiometry of 2 NADH consumed for each O₂ molecule and in the production of H₂O. e) The reaction is completely blocked by cyanide, superoxide dismutase (EC 1.15.1.1) and (excess) catalase (EC 1.11.1.6), but not by antimycin A or azide. This peroxidase-mediated O₂-uptake might interfere with respiratory measurements. In experiments with isolated mitochondria this interference can be prevented by the addition of catalase to the reaction mixture. The use of high concentrations of hydroxamate is not allowed because of inhibitory effects on the cytochrome pathway. In intact callus tissue hydroxamates only stimulate O₂-uptake in the presence of exogenous NADH. In vivo the peroxidase does not appear to function in O₂-uptake, probably because of its localization (at least partly in the cell wall) and/or its low affinity for NADH. The use of hydroxamates in the determination of cytochrome and alternative pathway activity is discussed.     

H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata

H₂O₂ production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H₂O₂ formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H₂O₂ generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O₂ respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O₂ uptake due to the formation of H₂O₂ cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H₂O₂. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.     

Cytochemical localization of hydrogen peroxide generating sites in the rat thyroid gland

Sites of H₂O₂ generation in lightly prefixed, intact thyroid follicles were studied by two cytochemical reactions: peroxidase-dependent DAB oxidation and cerium precipitation. In both cases reaction product accumulated on the apical surface of the follicle cell at the membrane-colloid interface. The former reaction was inhibited by the peroxidase inhibitor, aminotriazole; both reactions were blocked by the presence of catalase. NADH in the medium slightly increased the amount of cerium precipitation. The ferricyanide technique for oxidoreductase activity was also applied; reaction product again was associated with the apical surface. These results strongly imply that the follicle cells have a NADH oxidizing system generating H₂O₂ at the apical plasma membrane.     

Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase

Mitoxantrone (MH₂X), an anthraquinone-type anti-cancer agent used clinically in the treatment of human malignancies, is oxidatively activated by the peroxidase/H₂O₂ enzyme system. In contrast to the enzymatic mechanisms of drug oxidation, the chemical transformations of MH₂X are not well described. In this study, MH₂X metabolites, produced by the horseradish, lacto- or lignin peroxidase (respectively HRP, LPO and LIP)/H₂O₂ system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar mitoxantrone/H₂O₂ ratio, the efficacy of the enzyme-catalyzed oxidation of mitoxantrone decreased in the following order: LPO > HRP > LIP, which accorded with the decreasing size of the substrate access channel in the enzyme panel examined. In all cases, the central drug oxidation product was the redox-active cyclic metabolite, hexahydronaphtho-[2,3-f]-quinoxaline-7,12-dione (MH₂), previously identified in the urine of mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH₂ side-chains occurred, yielding the mono- and dicarboxylic acid derivatives respectively. Based on the available data a further MH₂ derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H₂O₂ concentrations, these novel MH₂ derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH₂ chromophore due to an oxidative cleavage of the 9,10-anthracenedione moiety. The novel metabolites extend the known sequence of peroxidase-induced mitoxantrone metabolism, and may contribute to the cytotoxic effects of the drug in vivo. Based on the structural features of the proposed MH₂ oxidation products we elaborate on various biochemical mechanisms, which extend the understanding of mitoxantrone’s pharmaceutical action and its clinical effectiveness with a particular focus on peroxidase-expressing solid tumors, such as breast carcinoma.     

Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

Oxidation of glutathione by peroxidase isoenzymes in fenugreek

During the germination of fenugreek (Trigonella foenum graecum L.) sulfhydryl groups rapidly declined in cotyledon and seedling axis, while peroxidase activity increased. Studies on purified isoenzymes showed that GSH was oxidized by the isoenzymes and was accomplished in presence of cofactors, Mn²⁺ and DCP along with H₂O₂ (0.01 mM). This reaction was found to be peroxidatic in nature. The oxidation was inhibited by catechol but was enhanced by malic acid.     

Isolation and characterization of wheat peroxidase isoenzyme B1

Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p-coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn²⁺ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o- and p- diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H₂O₂ from oxygen. Mn²⁺ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product.     

Purification, properties, and distribution of ascorbate peroxidase in legume root nodules

All aerobic biological systems, including N₂-fixing root nodules, are subject to O₂ toxicity that results from the formation of reactive intermediates such as H₂O₂ and free radicals of O₂. H₂O₂ may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN₃, CO, and C₂H₂ were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of ½ V_max was 70 micromolar for ascorbate and 3 micromolar for H₂O₂. The high affinity of ascorbate peroxidase for H₂O₂ indicates that this enzyme, rather than catalase, is responsible for most H₂O₂ removal outside of peroxisomes in root nodules.     

Effect of different phenols on the nadh-oxidation catalysed by a peroxidase from lupin

Cell wall-bound peroxidase (EC 1.11.1.7) from lupin (Lupinus albus) shows a transition from oxidase to peroxidase activity when it oxidizes NADH. The oxidase phase represents a lag period in the time course of the reaction. This phase is phenol-dependent and responsible for hydrogen peroxide formation. Guaiacol, an assay substrate, and p-coumaric, ferulic and sinapic acids, precursors of the cinnamyl alcohols used in the lignification process affect both the length of lag period and the rate of the peroxidase phase of NADH oxidation. The effect of different phenols on the time course of the reaction is related to the efficacy (V_max/K_m ratio) of the enzyme when it is acting on them as a peroxidese.     

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.