Oxidation of NADH by vanadium: kinetics, effects of ligands and role of H2O2 or O2.

The mechanism of oxidation of NADH by either vanadium(V) or vanadium(IV) was examined in the presence of reducing agents, complexing agents, and hydrogen peroxide. Reducing agents that stimulate the oxidation of NADH by V(V) include: a variety of cysteine analogues, glutathione, beta-mercaptoethanol, dithiothreitol, and ascorbate. Complexing agents which stimulate NADH oxidation by V(V) include cystine, glutathione disulfide, and dehydroascorbate. Vanadium(IV)-dependent systems which oxidize NADH include combinations of V(IV) with cysteine or air alone. Combination of either V(V) or V(IV) with hydrogen peroxide leads to NADH oxidation. Based on kinetic analysis and the use of the diagnostic inhibitors--superoxide dismutase, catalase, albumin, mannitol, ethanol, and anaerobic conditions--we have assigned two major mechanisms of NADH oxidation. One is the previously reported mechanism which involves V(V)-superoxide as the NADH oxidant. This reaction is inhibited by superoxide dismutase and anaerobic conditions but not by catalase or ethanol. This reaction is observed for V(V) in the presence of reducing agents and complexing agents. The second reaction mechanism operates when V(IV) comes in contact with hydrogen peroxide and involves V(III)-superoxide as the NADH oxidant. This reaction is inhibited by catalase (if unligated hydrogen peroxide is an intermediate) and superoxide dismutase but not anaerobic conditions or ethanol. This mechanism is observed for reactions of V(IV) with air or hydrogen peroxide.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Modulation of catalase peroxidatic and catalatic activity by nitric oxide

Previously, we found that catalase enhanced the protection afforded by superoxide dismutase to Escherichia coli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al., Arch. Biochem. Biophys. 316:327-334, 1995). Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxide dismutase. We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide. Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406 to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosyl complex. Catalase consumed more nitric oxide upon the addition of hydrogen peroxide. Conversely, micromolar concentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide. Catalase pretreated with nitric oxide and hydrogen peroxide regained full activity after dialysis. Our results suggest that catalase can slowly consume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide. The protective effects of catalase in combination with superoxide dismutase may result from two actions; reducing peroxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts with superoxide dismutase to form additional superoxide.     

Azo reduction of methyl red by neuronal nitric oxide synthase: the important role of FMN in catalysis

Nitric oxide synthase (NOS) is composed of an oxygenase domain and a reductase domain. The reductase domain has NADPH, FAD, and FMN binding sites. Wild-type nNOS reduced the azo bond of methyl red with a turnover number of approximately 130 min(-1) in the presence of Ca(2+)/calmodulin (CaM) and NADPH under anaerobic conditions. Diphenyleneiodonium chloride (DPI), a flavin/NADPH binding inhibitor, completely inhibited azo reduction. The omission of Ca(2+)/CaM from the reaction system decreased the activity to 5%. The rate of the azo reduction with an FMN-deficient mutant was also 5% that of the wild type. NADPH oxidation rates for the wild-type and mutant enzymes were well coupled with azo reduction. Thus, we suggest that electrons delivered from the FMN of the nNOS enzyme reduce the azo bond of methyl red and that this reductase activity is controlled by Ca(2+)/CaM.     

Interaction of riboflavin and hemoproteins with organic free radicals and superoxide anions generated in the ultrasound field

The reduction of ferricytochrome c to the ferro form in aqueous alcohol solutions in air by the action of ultrasound and the complete inhibition of this process in the presence of superoxide dismutase indicate the generation of superoxide anions. Further exposure to the ultrasonic field leads to a reverse process of oxidation of the cytochrome c ferro form to the ferri form by hydrogen peroxides and organic peroxides. The addition of catalase protects the cytochrome c ferro form from oxidation to the ferri form. The oxidized form of riboflavin effectively interacts with organic free radicals and superoxide anions to produce a leuko form, which is easily oxidized by air oxygen or the ferri forms of hemoglobin and cyt c to form riboflavin and hydrogen peroxide or the ferro forms of heme-containing proteins, respectively. The recurrence of redox reactions in the presence of riboflavin, organic free radicals, and O2 and the ferri forms of heme-containing proteins suggests that riboflavin can play a role of an antioxidant in the organism. It is supposed that, due to interaction with superoxide anions, riboflavin stabilizes the NO level in the organism under conditions of increased superoxide anion generation and (or) decreased superoxide dismutase activity. A possible role of riboflavin in the modulation of toxic and signal pathways of nitrogen oxide is discussed.     

Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain

The neuronal NO synthase (nNOS) flavin domain, which has similar redox properties to those of NADPH-cytochrome P450 reductase (P450R), contains binding sites for calmodulin, FAD, FMN, and NADPH. The aim of this study is to elucidate the mechanism of activation of the flavin domain by calcium/calmodulin (Ca(2+)/CaM). In this study, we used the recombinant nNOS flavin domains, which include or delete the calmodulin (CaM)-binding site. The air-stable semiquinone of the nNOS flavin domains showed similar redox properties to the corresponding FAD-FMNH(&z.ccirf;) of P450R. In the absence or presence of Ca(2+)/CaM, the rates of reduction of an FAD-FMN pair by NADPH have been investigated at different wavelengths, 457, 504 and 590 nm by using a stopped-flow technique and a rapid scan spectrophotometry. The reduction of the oxidized enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron equivalent mechanisms, and the formation of semiquinone (increase of absorbance at 590 nm) was significantly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form of the enzyme was also rapidly reduced by NADPH. The results suggest that an intramolecular one-electron transfer between the two flavins is activated by the binding of Ca(2+)/CaM. The F(1)H(2), which is the fully reduced form of the air-stable semiquinone, can donate one electron to the electron acceptor, cytochrome c. The proposed mechanism of activation by Ca(2+)/CaM complex is discussed on the basis of that provided by P450R.     

Important role of tetrahydrobiopterin in no complex formation and interdomain electron transfer in neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is composed of a heme oxygenase domain and a flavin-bound reductase domain. Ca(2+)/calmodulin (CaM) is essential for interdomain electron transfer during catalysis, whereas the role of the catalytically important cofactor, tetrahydrobiopterin (H4B) remains elusive. The product NO appears to bind to the heme and works as a feedback inhibitor. The present study shows that the Fe(3+)-NO complex is reduced to the Fe(2+)-NO complex by NADPH in the presence of both l-Arg and H4B even in the absence of Ca(2+)/CaM. The complex could not be fully reduced in the absence of H4B under any circumstances. However, dihydrobiopterin and N(G)-hydroxy-l-Arg could be substituted for H4B and l-Arg, respectively. No direct correlation could be found between redox potentials of the nNOS heme and the observed reduction of the Fe(3+)-NO complex. Thus, our data indicate the importance of the pterin binding to the active site structure during the reduction of the NO-heme complex by NADPH during catalytic turnover.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.     

Human neuronal nitric oxide synthase can catalyze one-electron reduction of adriamycin: role of flavin domain

We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca(2+)/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca(2+)/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD-NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH*) nor the air-stable semiquinone (FAD-FMNH*) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH(2)) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca(2+)/CaM. Based on these findings, we propose a role for the FAD-FMN pair in the one-electron reduction of Adr.     

Generation of hydrogen peroxide in resting and activated platelets.

The production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3-amino-1,2,4-triazole (AT) or N-ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.     

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reaction mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both .O2- and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H2O2 and .O2- are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (less than 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal chelating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (less than 5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (less than 10%) inhibition, but inhibited by 30% in the presence of catalase + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalase + superoxide dismutase + DETAPAC.     

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: possible implications for toxicity.

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H₂O₂ measured, but no H₂O₂ was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H₂O₂ production were enhanced by nitrofurantoin under anaerobic (N₂) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some $\text{in vitro}$ metabolic similarities with the lung-toxic herbicide, paraquat.     

NADH oxidase activity of indoleamine 2,3-dioxygenase

The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme.     

Storage of solar energy by production of hydrogen peroxide by the blue-green alga Anacystis nidulans R2: stimulation by azide

The production of hydrogen peroxide by Anacystis nidulans R2 in presence of methyl viologen occurs by using the redox power from water promoted by the photosystems of the blue-green alga. Thus, in the presence of the photosynthetic inhibitor DCMU or in the dark, H(2)O(2) production does not take place. In cells permeabilized with lysozyme, the addition of ionophores, which is expected to increase the electron flow, produces only a small increase to initial velocity of hydrogen peroxide production. On the other hand, in nonpermeabilized cells, the addition of superoxide dismutase increases the initial velocity of hydrogen peroxide production, but the net amount accumulated by the system is very low because of posterior decomposition. Preincubation of cells with azide, which inhibits the catalase, prevents the decomposition, thereby increasing drastically the amount of hydrogen peroxide accumulated by the system after a few hours. Hence, H(2)O(2) production appears to be limited mainly because of decomposition by catalase activity rather than by the photosynthetic electron flow rate or the diffusion of products through the cell wall. The net production of hydrogen peroxide by the system was enhanced severalfold by treatment with azide. If one takes into account the use of hydrogen peroxide as fuel due to the large amount of energy released in its dismutation, the photosystem can be a useful tool in the storage of solar energy.     

Oxidative Signals in Tobacco Increase Cytosolic Calcium

Tobacco (Nicotiana plumbaginifolia) seedlings genetically transformed to express apoaequorin were incubated in h-coelenterazine to reconstitute the calcium-sensitive luminescent protein aequorin. Treatment of these seedlings with hydrogen peroxide resulted in a transient burst of calcium-dependent luminescence lasting several minutes. Even though the hydrogen peroxide stimulus was persistent, the change in cytosolic free calcium concentration ([Ca2+]cyt) was transient, suggesting the presence of a refractory period. When seedlings were pretreated with hydrogen peroxide, there was no increase in [Ca2+]cyt upon a second application, which confirmed the refractory character of the response. Only when the two treatments were separated by 4 to 8 hr was full responsiveness recovered. However, treatment with hydrogen peroxide did not inhibit mobilization of [Ca2+]cyt induced by either cold shock or touching, suggesting that these three signals mobilize different pools of intracellular calcium. To examine whether [Ca2+]cyt is regulated by the redox state of the cytoplasm, we pretreated seedlings with buthionine sulfoximine (to modify cellular glutathione levels) and inhibitors of ascorbate peroxidase. These inhibitors modify the hydrogen peroxide-induced transients in [Ca2+]cyt, which is consistent with their effects on the cellular prooxidant/antioxidant ratio. Treatment with hydrogen peroxide that elicited [Ca2+]cyt increases also brought about a reduction in superoxide dismutase enzyme activity. This reduction could be reversed by treatment with the calcium channel blocker lanthanum. This indicates that there is a role for calcium in plant responses to oxidative stress.     

Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants

* Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Treatment with ABA or H(2)O(2) led to significant increases in the concentration of cytosolic Ca(2+) in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 (CaM1) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists. * Pretreatments with CaM antagonists almost completely inhibited ABA-induced H(2)O(2) production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA. * Our results suggest that Ca(2+)-CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca(2+)-CaM and H(2)O(2) plays a pivotal role in ABA signaling.     

Calorimetric studies of oxyhemoglobin ligand dissociation. III. Evidence for a hemoglobin-catalase-superoxide dismutase integrated system in the oxygen depletion by dithionite

Calorimetric studies of the effect of superoxide dismutase and/or catalase on the reduction of dioxygen into water by dithionite in oxyhemoglobin have been carried out and the results compared with those in red cell hemolysates. In the absence of the enzymes the stoichiometry (moles dithionite/mole dioxygen) is less than the value of 2:1 which was found previously in red cell hemolysates [Forlani et al., J. Inorg. Biochem. 20, 147-155 (1984)]. In the presence of either superoxide dismutase or catalase alone the stoichiometry increases but is still less than 2:1. In the presence of both enzymes the stoichiometry and the shape of the thermogram is that previously observed for hemolysates, suggesting the presence of a hemoglobin-catalase-superoxide dismutase integrated system. The absence of a calorimetric signal for hydrogen peroxide in the reduction of oxyhemoglobin in the presence of superoxide dismutase suggests a wider biological role of superoxide dismutase than previously thought.     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.