The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H(2)O(2). Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 x 10(7) M(-1) s(-1) for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.     

Hydrogen peroxide is involved in hamster sperm capacitation in vitro

We have investigated the possibility that the generation of hydrogen peroxide (H2O2) by spermatozoa plays a physiological role during capacitation. Capacitation is defined as the incubation period required for fertilization in mammals. Capacitation culminates in an exocytotic event, the acrosome reaction (AR). Mammalian sperm generate H2O2 during aerobic incubation and do not contain catalase, the enzyme that promotes scavenging of H2O2. In the present work we show that added catalase inhibited the AR, while glucose oxidase (GO), an enzyme that generates H2O2, accelerated the onset of the AR. Direct addition of H2O2 also stimulated the AR; catalase inhibited both the stimulation by GO and by H2O2. The onset of the AR was always preceded by the appearance of hyperactivated motility. The stimulation of the AR by H2O2 was manifest 1-2 h after the addition of H2O2. Catalase added at 3 h of incubation was less effective in inhibiting the AR than catalase added at the beginning. Incubation of sperm with catalase prevented the induction of the AR by the membrane-perturbing lipid, lysophosphatidyl choline. Taken together, these results suggest that H2O2 produced by hamster sperm plays a significant role during capacitation, possibly in membrane reorganization to facilitate the fusion that takes place during exocytosis of the acrosomal contents.     

Influence of Catalase Activity on Resistance of Coagulase-positive Staphylococci to Hydrogen Peroxide

Catalase activities of intact cells and cell-free extracts of coagulase-positive staphylococcal cultures 105B and 558D isolated from milk, culture 25042 from a clinical source, and Staphylococcus aureus 196E were determined at 32.2 C. Cultures were treated with 0.025 and 0.05% hydrogen peroxide at 37.8 and 54.4 C and without hydrogen peroxide at 54.4 C to determine the relationship between catalase activity and resistance to these treatments. The relationship held true for cultures 105B and 196E; culture 105B had the lowest catalase activity and lowest resistance to H(2)O(2) at 37.8 C, whereas S. aureus 196E possessed a high catalase activity and was most resistant at 37.8 C. Catalase activities of cell-free extracts of cultures 25042, 558, and 196E were similar, but resistance to H(2)O(2) at 37.8 C was greater for culture 196E. The lower resistance of culture 25042 was related to low catalase activities of whole cells of this culture, which were only one-third that of whole cells of culture 196E. Culture 558 was least resistant to heat treatment at 54.4 C and showed the greatest sensitivity to added H(2)O(2) at this temperature.     

Human serum catalase decreases endothelial cell injury from hydrogen peroxide.

Serum from normal human subjects contained variable amounts of catalase activity, which was inhibitable by heat, azide, trichloroacetic acid (TCA), or aminotriazole treatment. Serum also decreased hydrogen peroxide (H2O2) concentrations in vitro and H2O2-mediated injury to cultured endothelial cells. By comparison, heat-, azide-, TCA-, or aminotriazole-treated serum neither decreased H2O2 concentrations in vitro nor reduced H2O2-mediated damage to endothelial cells. We conclude that serum catalase activity can alter H2O2-dependent reactions. We speculate that variations in serum catalase activity may alter individual susceptibility to oxidant-mediated vascular disease or be a factor when added to test systems in vitro.     

Enzymatic oxidation of mercury vapor by erythrocytes

The formation of glutathione radicals, the evolution of nascent oxygen or the peroxidatic reaction with catalase complex I are considered as possible mechanisms for the oxidation of mercury vapor by red blood cells. To select among these, the uptake of atomic mercury by erythrocytes from different species was studied and related to their various activities of catalase (hydrogenperoxide : hydrogen-peroxide oxidoreductase, EC 1.11.1.6) and glutathione peroxidase (glutathione : hydrogen-peroxide oxidoreductase, EC 1.11.1.9). A slow and continuous infusion of diluted H2O2 was used to maintain steady concentrations of complex I. 1% red cell supsensions were found most suitable showing high rates of Hg uptake and yielding still enough cells for subsequent determinations. The results indicate that the oxidation of mercury depends upon the H2O2-generation rate and upon the specific acticity of red-cell catalase. The oxidation occurred in a range of the catalase-H2O2 reaction where the evolution of oxygen could be excluded. Compounds reacting with complex I were shown to be effective inhibitors of the mercury uptake. GSH-peroxidase did not participate in the oxidation but rather, was found to inhibit it by competing with catalase for hydrogen peroxide. These findings support the view that elemental mercury is oxidized in erythrocytes by a peroxidatic reaction with complex I only.     

A peroxide-induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis

In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA-binding protein MrgA. We have identified the P-type metal-transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon. Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region. Physiological studies implicate ZosA in Zn(II) uptake. ZosA functions together with two Zur-regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis. ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur-regulated transporters. A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation. ZosA is important for resistance to both H2O2 and the thiol-oxidizing agent diamide. This suggests that increased intracellular Zn(II) may protect thiols from oxidation. In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance. Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA. Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.     

Ascorbic acid potentiates the cytotoxicity of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin

The interaction of ascorbic acid with 5-methoxy-3,4-dehydroxanthomegnin, an 1,4-naphthoquinone, was investigated using the cytotoxic index for McCoy cells by neutral red assay. The synergistic effect was observed when such compounds were added simultaneously, most probably due to hydrogen peroxide being generated by ascorbate-driven 5-methoxy-3,4-dehydroxanthomegnin redox cycling. Incubation of cells in the presence of 5-methoxy-3,4-dehydroxanthomegnin/ascorbic acid/catalase, an enzyme that destroys H2O2, resulted in an increase of cell survival, reinforcing the involvement of hydrogen peroxide generated as an important oxidizing agent that kills McCoy cells.     

The function of catalase-bound NADPH

Catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) is of historical interest for having been the subject of some of the earliest investigations of enzymes. A feature of catalase that has been poorly understood for several decades, however, is the mechanism by which catalase remains active in the presence of its own substrate, hydrogen peroxide. We reported recently that catalase contains tightly bound NADPH. The present study with bovine and human catalase revealed that NADPH both prevents and reverses the accumulation of compound II, an inactive form of catalase that is generated slowly when catalase is exposed to hydrogen peroxide. Since the effect of NADPH occurs even at NADPH concentrations below 0.1 microM, the protective mechanism is likely to operate in vivo. This discovery of the role of catalase-bound NADPH brings a unity to the concept of two different mechanisms for disposing of hydrogen peroxide (catalase and the glutathione reductase/peroxidase pathway) by revealing that both mechanisms are dependent on NADPH.     

Achacin induces cell death in HeLa cells through two different mechanisms

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.     

The Positive Effect of Exogenous Hemin on a Resistance of Strict Anaerobic Archaeon Methanobrevibacter arboriphilus to Oxidative Stresses

Methanogenic archaeon Methanobrevibacter arboriphilus (strains AZ and DH1), which is a strict anaerobic microorganism not able to synthesize heme, possessed a very high catalase activity in the presence of 20-50?μM hemin in a growth medium. We investigated the effect of various oxidative stresses (hydrogen peroxide and oxygenation) on M. arboriphilus cells grown on the standard nutrient medium supplemented with 0.1?% yeast extract, and on the same medium supplemented with hemin. It was demonstrated that 30?μM hemin had a very significant positive effect on the resistance of M. arboriphilus strains to H(2)O(2) and O(2) stresses because of 30- to 40-fold increase of heme catalase activity. Thus, hydrogen peroxide (0.6-1.2?mM) or oxygen (3-5?%) had a strong negative impact on low-catalase cultures grown in the hemin-free standard medium, whereas the presence of 30?μM hemin in the medium results in a high yield of biomass even under conditions of four times stronger H(2)O(2) and two times stronger O(2) stresses. The intracellular catalase activity reached a high level in 30-60?min after hemin was added to the nutrient medium, but the activity already increased about 5-7-fold in 6?min after hemin addition. Our experimental data suggest that exogenous hemin provides an effective antioxidative defense in representatives of the genus Methanobrevibacter, specially playing an important role in the puromycin-insensitive formation of the active heme-containing catalase from presynthesized apoenzyme and heme.     

Hydrogen peroxide content and catalase activity at inoculation with root tubercle bacteria of pea seedlings with the various nodulation ability

Hydrogen peroxide (H2O2) content and catalase activity were studied in pea (Pisum sativum L.) seedlings with normal (cultivar Marat) and disrupted (pea mutants) process of nodulation, which were inoculated with the nitrogen-fixing bacterium Rhizobium leguminosarum strain CIAM 1026. Differences in hydrogen peroxide content and catalase activity in pea seedlings with different ability for nodulation, which were inoculated with rhizobia, were found. It was assumed that H2O2 and catalase are involved in defensive and regulatory mechanisms in the host plant.     

Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide

Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-cis-(1R,2S)-1,2-dihydroxy-1, 2-dihydronaphthalene with consumption of O2 and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O2 utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O2 was detected as hydrogen peroxide. The rate of benzene-dependent O2 consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O2 consumption by NDO. Detailed experiments showed that the total amount of O2 consumed and the rate of benzene-induced O2 consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISPNAP) of NDO. Kinetic studies showed that ISPNAP was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISPNAP gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISPNAP containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O2 consumption and as an inactivator of ISPNAP. It is proposed that the irreversible inactivation of ISPNAP occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (. OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [14C]benzene was used as the substrate, cis-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O2 consumption and NADH oxidation.     

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.     

Role of catalase and superoxide dismutase in the yeast Saccharomyces cerevisiae response to hydrogen peroxide in exponential phase of growth

The role of catalase and superoxide dismutase (SOD) in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide in the middle-exponential phase has been investigated. It was shown that cell survival is significantly decreased after yeast exposure to hydrogen peroxide in the strains defective in cytosolic or peroxisomal catalases. Treatment of the wild-type cells with 0.5 mM H2O2 for 30 min causes an increase in the activity of catalase and superoxide dismutase, but the effect was not observed in all strains investigated. It was also shown that hydrogen peroxide leads to an increase in the activities of both catalases and Cu,Zn-containing SOD. The effect was cancelled by cycloheximide, an inhibitor of protein synthesis.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.     

Superoxide removal and radiation protection in bacteria

Previous work with procaryotic cells has identified one kind of lethal damage from ionizing radiation which occurs only within a specific range of low O2 concentrations, about 10(-6) to 10(-4) M. Within this range, protection can occur in three ways: through the enzymatic decomposition of hydrogen peroxide (H2O2) by added catalase, through the enzymatic degradation of superoxide anion radicals (.O2-) by added superoxide dismutase (SOD), and through scavenging hydroxyl radicals (.OH) by various additives. These results indicate that three radiolytic products, H2O2, .OH, and .O2- (and/or the conjugate acid, the perhydroxyl radical, .HO2) are involved in this single kind of radiation-induced damage. Although the radiolytic productions of H2O2 and .O2- are strongly enhanced in higher O2 concentrations, neither enzyme protects when these air-equilibrated bacteria are irradiated. These experiments address this apparent contradiction and focus on the specific issue of why the addition of SOD protects at low but not at high O2 concentrations. We propose that, at a given O2 concentration, .O2- (and/or .HO2) may either react (with some cellular component?) to cause damage or react (with itself) to form hydrogen peroxide (H2O2). The specific O2 concentration during irradiation would determine the relative rates of these competing reactions and therefore the O2 concentration itself would establish whether or not we will observe damage from .O2-.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Effects of Some Chemicals on the Oxygen Evolution and Oxygen Uptake in Isolated Wheat Chloroplasts Irradiated without the Addition of an Oxidant

The oxygen exchange, obtained when isolated chloroplasts of Triticum aestivum, wheat, are irradiated without the addition of a Hill oxidant has been investigated using an oxygen electrode. Ascorbate, catalase, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone(DBMIB), diethyldithio-carbamate (DEDT), dichlorophenylmethylurea (DCMU), and potassium cyanide were added to the Chloroplasts in order to investigate the oxygen exchange. At least two oxygen uptake reactions, one sensitive to catalase and one catalase-insensitive, appeared upon irradiation. Hydrogen peroxide was the product of the oxygen uptake in the former process, and water was the reductant. The formation of hydrogen peroxide was probably associated with photosystem I. The other oxygen consuming reaction was found to be insensitive to both catalase and potassium cyanide. After the chloroplasts had been treated with DCMU, it was possible to show that the catalase-insensitive oxygen uptake was localized in photosystem I, and that a cyclic electron transport system or some endogenous reductant (-s) acted in the oxygen uptake. Addition of ascorbate or DEDT to the chloroplasts led to an enhanced oxygen uptake in 710 nm light. This was probably due to the effect of these compounds on the superoxide radical ion formed in photosystem I. The stimulated oxygen uptake was only weakly affected by catalase, indicating that hydrogen peroxide was not a product of this oxygen uptake. Addition of DEDT and potassium cyanide inhibited (strongly respectively weakly) the oxygen uptake when photosystem II was functioning. The effect of these compounds was probably due to an inhibition of the electron transport at the plastocyanin. DBMIB inhibited the oxygen uptake reactions and the cooperation between the two photosystems. The cooperation between the photosystems was also studied in DCMU-treated chloroplasts. The reactions in photosystem II, measured as oxygen evolution, were more inhibited than the coupling between the photosystems. The oxygen “gush” appearing upon irradiation in light of 650 nm was not affected by a DBMIB-treatment, showing that the oxygen evolution was due to the reduction of plastoquinone. The reoxidation in the dark of the plastoquinone pool was stimulated by DBMIB and potassium cyanide indicating that an oxygen uptake could be associated with plastoquinone. The sites of interaction of oxygen with the electron transport pathways in chloroplasts, and the different reductants for the oxygen consuming reactions are discussed.