Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Protective Properties of Tin- and Manganese-Centered Porphyrins Against Hydrogen Peroxide-Mediated Injury in Rat Astroglial Cells

Abstract: Tin-mesoporphyrin (tin-mp), a potent inhibitor of heme oxygenase, and manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a potent superoxide dismutase mimetic, reduced H₂O₂ toxicity in cultures of transformed rat astroglial cells if added 30 min before, or at the same time as, H₂O₂. Reduced toxicity was not observed if treatment was delayed for 60 min, the time by which H₂O₂ was essentially eliminated from cultures. Coadministration of tin-mp and MnTMPyP did not increase protection over either compound administered individually. Tin-mp, but not MnTMPyP, was stable in culture. MnCl₂ was not protective, suggesting that protection by MnTMPyP was not dependent on manganous ion, a by-product of MnTMPyP breakdown. Protection by tin-mp and MnTMPyP was not associated with metalloporphyrin-mediated induction of heme oxygenase-1 or with changes in heme oxygenase-2 on western blots. Whereas protective concentrations of tin-mp did not have superoxide dismutase-mimetic properties in vitro, protective concentrations of MnTMPyP partially inhibited heme oxygenase. The data support the hypothesis that heme oxygenase inhibition is protective against acute oxidative injury.     

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H₂O₂ at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H₂O₂ production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H₂O₂. Importantly, we demonstrate that a substantial portion of H₂O₂ production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Abstract

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O₂^??) and hydrogen peroxide (H₂O₂), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H₂O₂-eliminating enzyme, catalase, but not in the presence of an O₂^??-eliminating enzyme, superoxide dismutase. Treatment with H₂O₂ itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H₂O₂ treatment decreased in H₂O₂-resistant cells. Although both UVB and H₂O₂ are known to induce DNA damage, other DNA damaging agents, like γ-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H₂O₂ was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H₂O₂), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H₂O₂ together did not protect them from the H₂O₂-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H₂O₂ toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats

Hyperhomocysteinemia (HHcy) is a critical independent risk factor for cardiovascular diseases. However, to date, no satisfactory strategies to prevent HHcy exist. Since homocysteine (Hcy) and endogenous H₂S are both metabolites of sulfur-containing amino acids, we aimed to investigate whether a metabolic product of Hcy and H₂S, may antagonize in part the cardiovascular effects of Hcy. In the HHcy rat model injected subcutaneously with Hcy for 3 weeks, H₂S levels and the H₂S-generating enzyme cystathionine γ lyase (CSE) activity in the myocardium were decreased. The intraperitoneal injection of H₂S gas saturation solution significantly reduced plasma total Hcy (tHcy) concentration and decreased lipid peroxidation formation (i.e., lowered manodialdehyde and conjugated diene levels in myocardia and plasma). The activities of myocardial mitochondrial respiratory enzymes succinate dehydrogenase, cytochrome oxidase, and manganese superoxide dismutase, related to reactive oxygen species metabolism, were significantly dysfunctional in HHcy rats. The H₂S administration restored the level of enzyme activities and accelerated the scavenging of H₂O₂ and superoxide anion generated by Hcy in isolated mitochondria. The H₂S treatment also inhibited the expression of glucose-regulated protein 78, a marker of endoplasmic reticulum (ER) stress, induced by Hcy in vivo and in vitro. Thus, HHcy impaired the myocardial CSE/H₂S pathway, and the administration of H₂S protected the myocardium from oxidative and ER stress induced by HHcy, which suggests that an endogenous metabolic balance of sulfur-containing amino acids may be a novel strategy for treatment of HHcy.     

Hyperoxia increases oxygen radical production in rat lung homogenates

Lung damage during hyperoxia has been postulated to be due to increased rates of local organ oxygen radical production. Lung homogenate respiration was inhibited with cyanide, and residual respiration was used as an indicator of electron diversion to O₂^? and H₂O₂. Cyanide-resistant respiration in lung homogenates, supplemented with 1 mm NADH, increased linearly with oxygen tension, and accounted for 7% of total respiration in air and for 17% of total respiration when homogenates were incubated in 80% oxygen. Exposure of rats to 85% oxygen for 7 days induces tolerance to the lethal effects of 100% oxygen. Rats which previously breathed 85% oxygen for 7 days had a greater CN^?-resistant respiration than control rats. This implies that adaptation to hyperoxia does not include decreased lung tissue oxygen radical production as indicated by CN^?-resistant respiration. One possible explanation for the increased CN^?-resistant respiration in oxygen tolerant rat lungs is that they contain increased cell mass. Lung homogenates of rats exposed to 85% oxygen for 7 days also had 2.5 times greater thiobarbituric acid positive material than controls, indicating that increased lung lipid peroxidation occurs as a consequence of hyperoxia. Incubation of normal rat lung homogenates under hyperoxic conditions also acutely increased lipid peroxidation, which could be inhibited by both superoxide dismutase and catalase. This confirms that hyperoxia enhances cellular production of O₂^? and H₂O₂ and implies an essential role for both O₂^? and H₂O₂ in hyperoxic lung damage.     

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated.It was found that:

1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide.

2) with or without cyanide in the incubation medium, LG omitted, Mn⁺⁺, in the presence of NADPH induced superoxide anion (O¯₂) production, as evidenced by oxygen consumption and H₂O₂ production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O¯₂ scavenger).

3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occured only in the presence of Mn⁺⁺, and both were inhibited by superoxide dismutase.

4) Cyanide-resistant NADPH oxidation by LG generated H₂O₂, was inhibited by H₂O₂ and was not modified by «active catalase. The ratio of cyanide-resistant NADPH oxidation/O₂ uptake was 1 up to 1.25 mM NADPH, and increased above this concentration.

5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H₂O₂. The ratio of cyanide-sensitive NADPH oxidation/O₂ uptake was 2.

It was concluded that after initiation by O¯₂, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O¯₂-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H₂O₂ and O¯₂ occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H₂O₂ takes place.     

Redox state of cytochrome C in the presence of the 6-hydroxydopamine/oxygen couple: Oscillations dependent on the presence of hydrogen peroxide or superoxide

The reduction of ferricytochrome c in the presence of 6-hydroxydopamine/O₂ mixtures was examined under various reaction conditions. As the autoxidation of 6-hydroxy-dopamine progressed to completion, there were fluctuations in the net redox reactivity between reducing and oxidizing steady states. This was reflected in a sequence of damped oscillations in the redox state of cytochrome c. Corresponding to the time when 6-hydroxydopamine was 75–100% exhausted, reoxidation of the ferrocytochrome c occurred (prevented by catalase or catalase plus Superoxide dismutase). After the H₂O₂, in turn, was mostly consumed, the next phase commenced in which the cytochrome c became reduced for a second time. This reductive phase was 52% inhibited by superoxide dismutase. In the subsequent and final phase of the process, a progressive oxidation of cytochrome c lasting at least 24 h was observed. Of the initial reduction of ferricytochrome c, at most 37% can be attributed to direct reduction by 6-hydroxydopamine or its semiquinone. This initial net reduction of cytochrome c was inhibited 51% by superoxide dismutase and 41% by catalase. However, since either catalase or superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine by at least as much as it slowed the reduction of cytochrome c, their effects in slowing the reduction of cytochrome c resulted largely from the decreased production of those free radicals which reduce ferricytochrome c, and only in part from accelerated removal. Elimination of the actions of transition metal ions (whether by passage of the buffer solutions through Chelex 100 resins or by addition of desferrioxamine to the reaction medium) slowed both the reoxidation and rereduction by up to 96%. Addition of mannitol decreased the rate of the first reoxidation by 25% and increased the rate of the rereduction by 7%. In general, the oscillations are explicable in terms of changes in the steady state levels of O₂ and H₂O₂, with metal ions playing a major role and hydroxyl radicals a minor role in both the reoxidation and rereduction.     

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.     

Purification and Characterization of a Nylon-Degrading Enzyme

A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H₂O₂ but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, α-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.     

Exogenous Hydrogen Peroxide Changes Antioxidant Enzyme Activity and Protects Ultrastructure in Leaves of Two Cucumber Ecotypes Under Osmotic Stress

Cucumber (Cucumis sativus L.) varieties cv. Jinchun no. 4 (a North China ecotype) and cv. Lvfeng no. 6 (a South China ecotype) were cultivated to explore the effects of osmotic stress on the ultrastructure of chloroplasts and mitochondria, as well as to assess the possible protective effect of exogenous hydrogen peroxide (H₂O₂). Under osmotic stress induced by 10% polyethylene glycol 6000, 84.3% of the chloroplasts in Jinchun no. 4 were abnormal, whereas 88.6% were abnormal in Lvfeng no. 6. Abnormal mitochondria occurred in these two strains at rates of 78.5 and 87.1%, respectively. The stress condition disintegrated the membranes of most chloroplasts and mitochondria in the leaf cells of both cucumber ecotypes, and it also increased the malondialdehyde (MDA) content. We subjected the two cultivars to a combined treatment with H₂O₂ and osmotic stress and made the following observations: (1) Abnormal chloroplasts occurred at rates of 25.7 and 28.6%, and abnormal mitochondria were observed at rates of 22.9 and 32.8%, respectively. (2) Most of the investigated membranes were well organized in leaves of Jinchun no. 4 and Lvfeng no. 6, and the levels of endogenous H₂O₂, superoxide anion, and MDA were lower. Osmotic stress and exogenous H₂O₂ both increased the activities of antioxidative enzymes such as manganese superoxide dismutase, glutathione peroxidase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and the antioxidants ascorbate and reduced glutathione. The combined effect of osmotic stress and exogenous H₂O₂ resulted in the highest antioxidant activities in both cucumber ecotypes. We propose that exogenous H₂O₂ increases antioxidant activity in cucumber leaves and thereby decreases lipid peroxidation to some extent, thus protecting the ultrastructure of most chloroplasts and mitochondria under osmotic stress.     

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD⁺ or NADH at about the same operating redox potential as the NADH/NAD⁺ pool and comprise the NADH/NAD⁺ isopotential enzyme group. Complex I (specifically the flavin, site I_F) is often regarded as the major source of matrix superoxide/H₂O₂ production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H₂O₂ production. To differentiate the superoxide/H₂O₂-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H₂O₂ production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H₂O₂ production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)⁺ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H₂O₂ at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H₂O₂ was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site I_F of complex I. Depending on the substrates present, the dominant sites of superoxide/H₂O₂ production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.     

Hydrogen peroxide formation of polymorphonuclear leukocytes stimulated with cytochalasin D

Possible cellular sites to form superoxide (O₂⁻) and H₂O₂ were studied in polymorphonuclear leukocytes stimulated with either cytochalasin D (CD) or bacteria. When the cells were stimulated with cytochalasin, the ratio of O₂⁻ to H₂O₂ appearing in the medium was about 2 (determined separately) and the appearance of H₂O₂ in the medium was inhibited by the addition of cytochrome c, a scavenger of O₂⁻. The inhibition was reversed by the addition of superoxide dismutase. The cells phagocytosing bacteria released O₂⁻ at the beginning but the release subsided corresponding to the increase in the direct release of H₂O₂. p-Chloromercuribenzenesulfonate, a non-permeant reagent, inhibited the H₂O₂ formation by the cells stimulated with CD, whereas it did not inhibit the formation by the phagocytosing cells. These observations supported a hypothesis that the cells stimulated with the cytochalasin release O₂⁻ from the cell surface, whereas the phagocytosing cells release H₂O₂ from an intracellular site, probably the phagosomes formed by the invagination of the plasma membrane. An electron microscopic finding that the vacuole-like structures found in the cytochalasin-treated cells have openings to outside, also supported the hypothesis. The particle-bound NADPH-oxidase activity of the cytochalasin-treated cells was several times higher than that of the resting cells and the activation was apparently parallel with the O₂⁻-releasing activity of the cells. No activation of the NADH-oxidase was observed.     

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

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

Red cell lysis induced by microorganisms as a case of superoxide- and hydrogen peroxide-dependent hemolysis mediated by oxyhemoglobin

Some bacteria, isolated from the blood of hospitalized patients, have been shown to hemolyze red blood cells through a mechanism which was dependent on the oxygenated state of intracellular hemoglobin, since transformation of hemoglobin into the CO-derivative inhibited the lysis. Hemolysis was also inhibited by superoxide dismutase and catalase, while only catalase prevented the formation of methemoglobin in experiments where isolated oxyhemoglobin was exposed to metabolizing bacteria. Production by bacteria of extracellular superoxide was demonstrated. It is suggested that hemolysis is due to interaction of O₂⁻ and/or H₂O₂ with intracellular hemoglobin and that some product of such interaction is the lytic agent.