Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Mechanism of H2O2 production in porcine thyroid cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H2O2-generating machinery.

Hydrogen peroxide (H2O2), which is required for thyroid hormone synthesis, has been believed to be produced at the apical cell surface of thyroid follicular cells. However, we recently found that plasma membrane from porcine thyroid exclusively generated superoxide anion (O2-) by employing a novel method for simultaneous determination of H2O2 and O2- with diacetyldeuterioheme-substituted horseradish peroxidase (diacetyl-HRP) as the trapping reagent [Nakamura, Y., Ohtaki, S., Makino, R., Tanaka, T., & Ishimura, Y. (1989) J. Biol. Chem. 264, 4759-4761]. The present study describes the mechanism of H2O2 production as analyzed by this new method. Incubation of cultured porcine follicular cells with ionomycin, a Ca-ionophore, caused an increase in oxygen uptake of about 80%. During enhanced respiration, the cells released H2O2 in an amount equivalent to the amount of oxygen consumed as judged by the formation of compound II of diacetyl-HRP, and H2O2 adduct of the peroxidase. No formation of compound III of the peroxidase, an O2- adduct, was detected during burst respiration. Thus, the intact cells exclusively released H2O2 to the outside of the cells. On the other hand, when the cell fragments from follicular cells were incubated with NADPH or NADH in the presence of Ca2+, the production of O2- was observed only during NADPH-dependent burst respiration, supporting our previous results that the plasma membrane exhibited NADPH-dependent O2(-)-generating activity. O2- production by the plasma membrane was further confirmed by analyses of the effects of superoxide dismutase (SOD) and catalase on the reaction. These results suggested that H2O2 is secondarily produced through the dismutation of O2-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase

Extracellular release of superoxide anion (O-2) and hydrogen peroxide (H2O2) during the respiratory burst of porcine and human neutrophils was studied by using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for these oxygen derivatives. The method permitted simultaneous measurement of oxygen consumption and formation of both O-2 and H2O2 in a single reaction mixture. When neutrophils were stimulated with phorbol myristate acetate in the presence of the heme-substituted peroxidase, a rapid accumulation of compound III, a complex of the enzyme with O-2, was observed accompanying an increase in oxygen consumption. During the process, amounts of compound III formed and oxygen consumed were stoichiometric, and no compound II, an indicator of H2O2 formation, was observed. These results establish that neutrophils stimulated with the phorbol ester produce exclusively O-2 as the primary oxygen metabolite and release it into the extracellular medium. When a limited amount of opsonized zymosan was used as the stimulus, compound III formation was also observed but it ceased at an early stage of oxygen consumption. When a sufficient amount of azide was included in the system, however, formation of compound II was noted in the later stage of oxygen consumption. The findings suggest that O-2, formed during phagocytosis, is converted to H2O2 within phagosomes and then diffuses out into the extracellular medium when its decomposition by catalase and/or peroxidases is blocked by azide.     

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.     

A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe(3+)), superoxide (O(-)(2)) is produced, and HRP-Fe(3+) is converted to compound III. Superoxide dismutase inhibited both the generation of O(-)(2) and the formation of compound III. In contrast, catalase inhibited only the generation of O(-)(2). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe(3+), NADH and oxygen. The generation of hydroxyl radicals was dependent upon O(-)(2) and H(2)O(2) produced by HRP-Fe(3+)-NADH. The reaction of compound III with H(2)O(2) caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe(3+)-NADH (but not K(+)O(-)(2) and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+…)O(2)(…)NAD(.) (but not free O(-)(2) or H(2)O(2)) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe(3+), compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O(-)(2) and hydroxyl radicals.     

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

Oxidation of melatonin and tryptophan by an HRP cycle involving compound III

We recently described that horseradish peroxidase (HRP) and myeloperoxidase (MPO) catalyze the oxidation of melatonin, forming the respective indole ring-opening product N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) (Biochem. Biophys. Res. Commun. 279, 657-662, 2001). Although the classic peroxidatic enzyme cycle is expected to participate in the oxidation of melatonin, the requirement of a low HRP:H(2)O(2) ratio suggested that other enzyme paths might also be operative. Here we followed the formation of AFMK under two experimental conditions: predominance of HRP compounds I and II or presence of compound III. Although the consumption of substrate is comparable under both conditions, AFMK is formed in significant amounts only when compound III predominates during the reaction. Using tryptophan as substrate, N- formyl-kynurenine is formed in the presence of compound III. Both, melatonin and tryptophan efficiently prevents the formation of p-670, the inactive form of HRP. Since superoxide dismutase (SOD) inhibits the production of AFMK, we proposed that compound III acts as a source of O(-*)(2) or participates directly in the reaction, as in the case of enzyme indoleamine 2,3-dioxygenase.     

Determinants of the phagosomal pH in neutrophils.

Phagosomes formed by neutrophils are much less acidic than those of other phagocytic cells. The defective acidification seen in neutrophils has been attributed to consumption of protons during the dismutation of superoxide, because a large, sustained acidification is unmasked when the cells are treated with inhibitors of the NADPH oxidase. Consumption of protons transported into the phagosome by dismutation would tightly couple the activities of the NADPH oxidase and the vacuolar type H(+)-pump (or V-ATPase). We tested the existence of the predicted coupling using microfluorimetry and digital imaging and found that the rate of superoxide generation was independent of the activity of the H(+)-pump. Moreover, we failed to detect the alkalinization predicted to develop through dismutation when the pump was inhibited. Instead, two other mechanisms were found to contribute to the inability of neutrophil phagosomes to acidify. First, the insertion of V-ATPases into the phagosomal membrane was found to be reduced when the oxidase is active. Second, the passive proton (equivalent) permeability of the phagosomal membrane increased when the oxidase was activated. The increased permeability cannot be entirely attributed to the conductive H(+) channels associated with the oxidase, since it is not eliminated by Zn(2+). We conclude that the NADPH oxidase controls the phagosomal pH by multiple mechanisms that include reduced proton delivery to the lumen, increased luminal proton consumption, and enhanced backflux (leak) into the cytosol.     

Mechanism of reaction of a suggested superoxide-dismutase mimic, Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The interaction of a recently developed intracellular superoxide dismutase analogue, Fe(II)-N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (Fe(II)-TPEN), with reactive oxygen species was investigated under in vitro conditions. The complex catalyzed the dismutation of enzyme- or radiolysis-generated superoxide with the production of H2O2; under steady-state conditions the equilibrium was strongly shifted toward Fe(III)-TPEN. Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radicals in a Fenton reaction. The oxidized Fe(III)-TPEN was readily reduced by ascorbate or glutathione. Given the capacity to produce hydroxyl radicals and the reaction with cellular reductants it seems unlikely that Fe-TPEN may find widespread use as an intracellular superoxide dismutase substitute.     

Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production

NADPH oxidases are major sources of superoxide (O2*-) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O2*- and H2O2. Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O2*- and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2*- and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2*- by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2*- production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMCs led to diminished basal H2O2 production, but did not affect O2*- or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O2*- and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2*- that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2*- production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O2*- scavengers that do not affect H2O2.     

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.     

Plasma Membrane Redox Enzyme Is Involved in the Synthesis of O2- and H2O2 by Phytophthora Elicitor-Stimulated Rose Cells

An elicitor prepared from the autoclaved cell walls of Phytophthora sp. induced O2- generation and H2O2 accumulation by cultured cells of Rosa damascena Mill. cv Gloire de Guilan. N,N-Diethyldithiocarbamate, a superoxide dismutase inhibitior, blocked H2O2 accumulation and caused a dramatic accumulation of O2- by elicitor-treated rose cells. In the absence of N,N-diethyldithiocarbamate no detectable O2- was accumulated. Diphenyleneiodonium, quinacrine, pyridine, and imidazole, inhibitors of the mammalian neutrophil NADPH oxidase responsible for the generation of O2- during phagocytosis, inhibited O2- generation by elicitor-treated rose cells. Diphenyleneiodonium also inhibited NADH-dependent O2- production by plasma membranes isolated from rose cells. None of the four compounds inhibited the peroxidase activity in the cell-suspension medium. These results demonstrate that elicitor-stimulated accumulation of H2O2 comes only from superoxide dismutase-catalyzed dismutation of O2-. The data are inconsistent with the hypothesis that the synthesis of O2- is catalyzed by extracellular peroxidase and suggest that the enzyme responsible for the synthesis of O2- by elicitor-treated rose cells might be similar to the mammalian neutrophil NADPH oxidase.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Mechanisms of protection of catalase by NADPH. Kinetics and stoichiometry.

NADPH is known to be tightly bound to mammalian catalase and to offset the ability of the substrate of catalase (H2O2) to convert the enzyme to an inactive state (compound II). In the process, the bound NADPH becomes NADP+ and is replaced by another molecule of NADPH. This protection is believed to occur through electron tunneling between NADPH on the surface of the catalase and the heme group within the enzyme. The present study provided additional support for the concept of an intermediate state of catalase, through which NADPH serves to prevent the formation (rather than increase the removal) of compound II. In contrast, the superoxide radical seemed to bypass the intermediate state since NADPH had very little ability to prevent the superoxide radical from converting catalase to compound II. Moreover, the rate of NADPH oxidation was several times the rate of compound II formation (in the absence of NADPH) under a variety of conditions. Very little NADPH oxidation occurred when NADPH was exposed to catalase, H2O2, or the superoxide radical separately. That the ratio exceeds 1 suggests that NADPH may protect catalase from oxidative damage through actions broader than merely preventing the formation of compound II.     

Generation of hydrogen peroxide during the oxidation of L-phenylalanine by Proteus mirabilis isolated membranes

In the process of L-phenylalanine oxidation by Proteus mirabilis cytoplasmic membrane, hydrogen peroxide was produced at a rate corresponding to 1-3 per cent of the total electron flow (30-110 nmoles min-1mg-1). Peroxide was estimated using a fluorimetric assay with horseradish peroxidase, or by anodic oxidation on a platinum electrode. When using the former method, superoxide dismutase decreased the apparent yield of peroxide, a fact suggesting that H2O2 was in part the dismutation product of superoxide radicals. However the superoxide dismutase effect could be an artefact due to the generation of some superoxide during the peroxidatic reaction in the assay. Adrenaline was the reagent used for the detection of superoxide. There was no significant emergence of superoxide as the result of phenylalanine oxidation by the membrane (specific activity lower than 1-2 nmoles min-1mg-1). Thus it seemed that superoxide was not an intermediate for the bulk of H2O2 formed in this system. According to the results, peroxide was probably formed at a stage of electron transport earlier than the cytochrome level. The membrane phenylalanine dehydrogenase could be a site where peroxide was evolved in these experiments.     

NADPH-dependent H2O2 generation catalyzed by thyroid plasma membranes. Studies with electron scavengers

Hog thyroid plasma membrane preparations containing a Ca2+-regulated NADPH-dependent H2O2-generating system were studied. The Ca2+-dependent reductase activities of ferricytochrome c, 2,6-dichloroindophenol, nitroblue tetrazolium, and potassium ferricyanide were tested and the effect of these scavengers on H2O2 formation, NADPH oxidation and O2 consumption were measured, with the following results. 1. Thyroid plasma membrane Ca2+-independent cytochrome c reduction was not catalyzed by the NADPH-dependent H2O2-generating system. This activity was superoxide-dismutase-insensitive. 2.Of the three other electron scavengers tested, only K3Fe(CN)6 was clearly, but partially reduced in a Ca2+-dependent manner. 3. Though the NADPH-dependent reduction of nitroblue tetrazolium was very low and superoxide-dismutase-insensitive, nitroblue tetrazolium inhibited O2 consumption, H2O2 formation and NADPH oxidation, indicating that nitroblue tetrazolium inhibits the H2O2-generating system. We conclude that the thyroid plasma membrane H2O2-generating system does not or liberate O2- and that Ca2+ controls the first step (NADPH oxidation) of the H2O2-generating system.     

Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor-induced Taxus chinensis cell cultures

After determining that hydrogen peroxide (H2O2) accumulation induced by a fungal elicitor from Aspergillus niger was from the superoxide dismutase-catalyzed dismutation of superoxide radical, the site of H2O2 generation in cell suspension cultures of Taxus chinensis was studied. The results showed that 90% and 10% of the elicitor-induced H2O2 accumulation respectively appeared in intracellular and extracellular fractions of cells, and that the elicitor-induced H2O2 accumulation in protoplasts and plasma membranes was similar to that in intact cells, indicating that the site of H2O2 accumulation was plasma membranes but not in extracellular fraction of Taxus cells. The H2O2 forming enzyme was also investigated. The elicitor-induced H2O2 accumulation in intact cells was not changed by loss of apoplastic peroxidase (POD) by the washing, and the H2O2 accumulation in plasma membranes was inhibited by the mammalian neutrophil NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), but was slightly affected by exogenous POD and its inhibitor. Furthermore, in plasma membranes, the H2O2 accumulation was more significantly enhanced by NADPH than by NADH, and the former was more obviously decreased by DPI than the latter. The present results show that NADPH oxidase in plasma membranes is involved in H2O2 accumulation in fungal elicitor-induced Taxus chinensis cell cultures.