Superoxide anion is the initial product in the hydrogen peroxide formation catalyzed by NADPH oxidase in porcine thyroid plasma membrane

The plasma membrane fraction from porcine thyroid is known to exhibit an NADPH-dependent production of hydrogen peroxide (H2O2), which is utilized for the oxidative biosynthesis of thyroid hormones catalyzed by thyroid peroxidase. The H2O2 formation is cyanide-insensitive, ATP-activatable, and Ca2+-dependent (Nakamura, Y., Ogihara, S., and Ohtaki, S. (1987) J. Biochem. (Tokyo) 102, 1121-1132). It remains unknown, however, whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of superoxide anion (O2-). We therefore attempted to analyze the mechanism of H2O2 formation by utilizing a new method for the simultaneous measurement of O2- and H2O2, in which diacetyldeuteroheme-substituted horseradish peroxidase was employed as the trapping agent for both oxygen metabolites. When NADPH was incubated with the membrane fraction in the presence of the heme-substituted peroxidase, a massive O2 consumption was observed together with the formation of compound III, and O2- adduct of the peroxidase. The amounts of compound III formed and O2 consumed were stoichiometric with each other, while formation of compound II, an indicative of H2O2, was not observed during the reaction. On the other hand, when an excess amount of superoxide dismutase was included in the reaction mixture, compound II was produced with complete suppression of the compound III formation. NADH minimally supported both O2 consumption and formation of compound III or II. These results indicate that the NADPH oxidase in the plasma membrane of thyroid produces O2- as the primary metabolite of O2 and hence that H2O2 required for the thyroid hormone synthesis provided through the dismutation of O2-.     

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).     

Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.     

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.     

Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 microM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67(phox) localized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67(phox) blocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.     

Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase,succinate dehydrogenase,and aspartate oxidase

Oxidative stress is created in aerobic organisms when molecular oxygen chemically oxidizes redox enzymes, forming superoxide (O2*-) and hydrogen peroxide (H2O2). Prior work identified several flavoenzymes from Escherichia coli that tend to autoxidize. Of these, fumarate reductase (Frd) is notable both for its high turnover number and for its production of substantial O2*- in addition to H2O2. We have sought to identify characteristics of Frd that predispose it to this behavior. The ability of excess succinate to block autoxidation and the inhibitory effect of lowering the flavin potential indicate that all detectable autoxidation occurs from its FAD site, rather than from iron-sulfur clusters or bound quinones. The flavin adenine dinucleotide (FAD) moiety of Frd is unusually solvent-exposed, as evidenced by its ability to bind sulfite, and this may make it more likely to react adventitiously with O2*-. The autoxidizing species is apparently fully reduced flavin rather than flavosemiquinone, since treatments that more fully reduce the enzyme do not slow its turnover number. They do, however, switch the major product from O2*- to H2O2. A similar effect is achieved by lowering the potential of the proximal [2Fe-2S] cluster. These data suggest that Frd releases O2*- into bulk solution if this cluster is available to sequester the semiquinone electron; otherwise, that electron is rapidly transferred to the nascent superoxide, and H2O2 is the product that leaves the active site. This model is supported by the behavior of "aspartate oxidase" (aspartate:fumarate oxidoreductase), an Frd homologue that lacks Fe-S clusters. Its dihydroflavin also reacts avidly with oxygen, and H2O2 is the predominant product. In contrast, succinate dehydrogenase, with high potential clusters, generates O2*- exclusively. The identities of enzyme autoxidation products are significant because O2*- and H2O2 damage cells in different ways.     

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide

Summary Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD⁺ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H₂O₂ production for every mole of NADH that was oxidized.Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of V^IV species by reduction of V^V of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.     

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.     

Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca2+/c-Abl signaling pathway

The importance of H₂O₂ as a cellular signaling molecule has been demonstrated in a number of cell types and pathways. Here we explore a positive feedback mechanism of H₂O₂-mediated regulation of the phagocyte respiratory burst NADPH oxidase (NOX2). H₂O₂ induced a dose-dependent stimulation of superoxide production in human neutrophils, as well as in K562 leukemia cells overexpressing NOX2 system components. Stimulation was abrogated by the addition of catalase, the extracellular Ca²⁺ chelator BAPTA, the T-type Ca²⁺ channel inhibitor mibefradil, the PKCδ inhibitor rottlerin, or the c-Abl nonreceptor tyrosine kinase inhibitor imatinib mesylate or by overexpression of a dominant-negative form of c-Abl. H₂O₂ induced phosphorylation of tyrosine 311 on PKCδ and this activating phosphorylation was blocked by treatment with rottlerin, imatinib mesylate, or BAPTA. Rac GTPase activation in response to H₂O₂ was abrogated by BAPTA, imatinib mesylate, or rottlerin. In conclusion, H₂O₂ stimulates NOX2-mediated superoxide generation in neutrophils and K562/NOX2 cells via a signaling pathway involving Ca²⁺ influx and c-Abl tyrosine kinase acting upstream of PKCδ. This positive feedback regulatory pathway has important implications for amplifying the innate immune response and contributing to oxidative stress in inflammatory disorders.     

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.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

Xanthine oxidase: a source of hydrogen peroxide in bovine thyroid glands.

Hydrogen peroxide produced by bovine thyroidal xanthine oxidase was found to yield protein bound iodine $\text{in vitro}$ in the presence of a thyroidal peroxidase. The thyroid metabolites, mono- and diiodotyramine, which have very potent inhibitory effects on thyroid monoamine oxidase have very little effect on thyroid xanthine oxidase below 1 mM concentration. Allopurinol and formycin B reduced the level of iodination of protein in thyroid tissue slices. These data suggest that thyroid xanthine oxidase may be an important source of the hydrogen peroxide required for thyroxine biosynthesis.     

The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

Vanadium catalyzed oxidation with hydrogen peroxide

Vanadium peroxides are known as very effective oxidants of different organic and inorganic substrates. In this short account reactivity, structural and mechanistic studies concerning the behaviour of peroxovanadates toward a number of different substrates are collected. Homogeneous and two-phase systems are presented, in addition, interesting synthetic results obtained with the use of ionic liquids as reaction media are also presented.     

Role of the plasma membrane ROS-generating NADPH oxidase in CD34+ progenitor cells preservation by hypoxia

Hypoxia favored the preservation of progenitor characteristics of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. This work aimed at studying the role of reactive oxygen species (ROS)-generating NADPH oxidase system regulated by hypoxia in ex vivo cultures of cord blood CD34⁺ cells. The results showed that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. Meanwhile the ROS generation was found to be inhibited by diphenyleneiodonium (the NADPH oxidase inhibitor), or N-acetylcysteine (the ROS scavenger). Accordingly NADPH oxidase mRNA and p67 protein levels decreased in hypoxia. The analysis of progenitor characteristics, including the proportion of cultured cells expressing the HSPCs marker CD34⁺CD38⁻, colony production ability of the colony-forming cells (CFCs), and the re-expansion capability of the cultured CD34⁺ cells, showed that either 5% pO₂ or reduced ROS favored preserving the characteristics of CD34⁺ progenitors, and promoted the expansion of CD34⁺CD38⁻ cells as well. The above results demonstrated that hypoxia effectively maintained biological characteristics of CD34⁺ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase.     

Stoichiometric cooperation of NADPH and hexobarbital in hepatic microsomes during the catalysis of hydrogen peroxide formation

The interaction of NADPH and hexobarbital during catalysis of microsomal mixed function oxidase-dependent hydrogen peroxide formation has been investigated in hepatic microsomes from phenobarbital-treated rabbits. The application of Job's method (25) of continuous variation revealed optimal conditions for the rate and extent of hydrogen peroxide formation when hexobarbital and NADPH were in equimolar amounts. The formation of a complex of 1 mol NADPH with cytochrome c-reductase and 1 mol hexobarbital with cytochrome P-450 seems to be responsible for limitation of hydrogen peroxide formation. Rate and extent of hydrogen peroxide formation are directly proportional to the amount of hexobarbital and NADPH present and are governed by the mass action equation in a manner similar to that reported for interaction of purified enzymes (G. T. Miwa, S. B. West, M. T. Huang, and A. H. Lu, 1979,J. Biol. Chem.254, 5695–5700). Depending on either the NADPH concentration maintained by a generating system or the hexobarbital concentration, the extent of hydrogen peroxide formation could be shown to be a function of either compound alone, as long as the other one is in excess. The question whether the formation of hydrogen peroxide depends on the availability of two independent one-electron transfer reactions forming O₂^? or of one simultaneous two-electron transfer forming O₂^2? might thus become rather a matter of association of substrate and cosubstrate to a catalytically active complex in which the substrate augments the availability of reducing equivalents.     

Co-activation of protein kinase C and NADPH oxidase in the plasma membrane of neutrophil cytoplasts

Enucleated, granule-free neutrophil cytoplasts, which in hypotonic media fully release cytosolic components and generate ghosts, have been used to study the cell localization of protein kinase C (PK-C). Treatment of cytoplasts with phorbol myristate acetate, a potent activator of neutrophil functions, triggers translocation of PK-C from the cytosol to the plasma membrane, with an activity recovery of 83 ± 16%. In the ghost fraction, PK-C catalyzes the phosphorylation of polypeptides with an apparent mol. wt. of 115K, 89K, 79K, 62K, 47K and 19K. From the plasma membrane PK-C can be extracted in an active form by Triton X-100 but not by EGTA. Translocation of PK-C is already evident at 5 sec and plateaus at about 50 sec. Activation of plasmalemmal, O₂⁻ generating NADPH oxidase by the phorbol ester is delayed by about 20 sec with respect to the activation of PK-C. Dose/response experiments show that the pattern of activation of O₂⁻ generation by cytoplasts strictly superimposes with the pattern of PK-C translocation.     

Modulation of bovine serum amine oxidase activity by hydrogen peroxide

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.