The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.

Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent. Oxygen, which protected against inactivation, also diminished the luminescence. Several other compounds which prevented the inactivation by H2O2 also modified the luminescence. Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it. These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme. The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light. Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies. This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid. In control experiments, boiled enzyme was inactive. This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2-. Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate.     

Relation between ESR-detectable Cu(II) and superoxide dismutase activity

The relation between ESR-detectable Cu(II) and Cu,Zn-superoxide dismutase activity was examined. The Cu(II) spin numbers per one unit of SOD were 6.26 X 10(12) (+/- 0.51 X 10(12] spins in several preparations of recombinant human Cu,Zn-SOD, native placental, and erythrocyte SOD. Measurement could be performed over a wide range of pH (4.0-10.0), preferably at temperatures below -40 degrees C. The data obtained by this method correlated well to the results obtained by the method of Fridovich et al. using the xanthine-xanthine oxidase system (correlation coefficient 0.995). The specific activity of SOD was proportional to the Cu(II) content measured by ESR, but not to the total Cu content measured by atomic absorption. This indicates that it is important to measure the Cu(II) content for determining Cu,Zn-SOD activity.     

The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme.

Bovine erythrocyte superoxide dismutase was slowly and irreversibly inactivated by hydrogen peroxide. The rate of this inactivation was directly dependent upon the concentrations of both H2O2 and of enzyme, and its second-order rate constant at pH 10.0 and 25 degrees was 6.7 M-1 sec-1. Inactivation was preceded by a bleaching due to rapid reduction of Cu2+ on the enzyme, and following this there was a gradual reappearance of a new absorption in the visible region, which was coincident with the loss of catalytic activity. Inactivation of the enzyme was pH-dependent and indicated an essential ionization whose pKa was approximately 10.2. Replacement of H2O by D2O raised this pKa but did not diminish the catalytic activity of superoxide dismutase, measured at pH 10.0. Several compounds, including xanthine, urate, formate, and azide, protected the enzyme against inactivation by H2O2. Alcohols and benzoate, which scavenge hydroxyl radical, did not protect. Compounds with special affinity for singlet oxygen were similarly ineffective. The data were interpreted in terms of the reduction of the enzyme-bound Cu2+ to Cu+, by H2O2, followed by a Fenton's type reaction of the Cu+ with additional H2O2. This would generate Cu2+-OH- or its ionized equivalent, Cu2+-O--, which could then oxidatively attack an adjacent histidine and thus inactivate the enzyme. Compounds which protected the enzyme could have done so by reacting with the bound oxidant, in competition with the adjacent histidine.     

Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs

Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O₂. Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O₂ to . Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of to H₂O₂, and the reductant dependent reduction of H₂O₂ to H₂O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.     

Effect of hydrogen peroxide on the iron-containing superoxide dismutase of Escherichia coli

The iron-containing superoxide dismutase from Escherichia coli is inactivated by H2O2 to a limit of approximately 90%. When corrected for the H2O2-resistant portion, this inactivation was first order with respect to residual activity and exhibited a pseudo-first-order rate constant of 0.066 min-1 at 25 degrees C in 0.24 mM H2O2 at pH 7.8. The superoxide dismutase activity remaining after treatment with H2O2 differed from the activity of the native enzyme with respect to heat stability, inhibition by azide, and inactivation by light in the presence of rose bengal and by N-bromosuccinimide. The native and the H2O2-modified enzymes were indistinguishable by electrophoresis on polyacrylamide gels. Inactivation of the enzyme by H2O2 was accompanied by loss of tryptophan and some loss of iron, but there was no detectable loss of histidine or of other amino acids. H2O2 treatment caused changes in the optical spectrum of the enzyme. Inactivation of the enzyme by H2O2 depends upon the iron at the active site. Thus, the apoenzyme and the manganese-substituted enzyme were unaffected by H2O2. We conclude that reaction of H2O2 with the iron at the active site generates a potent oxidant capable of attacking tryptophan residues. A mechanism is proposed.     

The stimulatory effect of negative air ions and hydrogen peroxide on the activity of superoxide dismutase

The activity of erythrocyte cytosolic superoxide dismutase from rat, bovine, man and duck was considerably increased when measured after preparation or incubation in media pretreated with negative air ions (mostly superoxide) from electroeffluvial ion generator. 0.5–1.0 μM H₂O₂ was found in incubation medium after treatment with air ions. The stimulatory effect of air ions on superoxide dismutase activity was mimicked by addition of 0.5–6 μM H₂O₂. The primary physicochemical mechanism of beneficial biological action of negative air ions is suggested to be related to the stimulation of superoxide dismutase activity by micromolar concentrations of H₂O₂.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Serum superoxide dismutase 3 (extracellular superoxide dismutase) activity is a sensitive indicator of Cu status in rats

Sensitivity of the assay for Cu,Zn superoxide dismutase 3 (SOD3), the predominant form of SOD in serum, can be increased, and interferences caused by low-molecular-weight substances in the serum can be reduced by conducting the assay at pH 10 with xanthine/xanthine oxidase and acetylated cytochrome c (cyt c) as superoxide generator and detector, respectively. Serum SOD3 activity was assayed under these conditions in an experiment where weanling, male rats were fed diets for 6 weeks containing 3, 5 and 15 mg Zn/kg with dietary Cu set at 0.3, 1.5 and 5 mg Cu/kg at each level of dietary Zn. Serum SOD3 responded to changes in dietary Cu but not to changes in dietary Zn. A second experiment compared serum SOD3 activity to traditional indices of Cu status in weanling, male and female rats after they were fed diets containing, nominally, 0, 1, 1.5, 2, 2.5, 3 and 6 mg Cu/kg for 6 weeks. Serum SOD3 activity was significantly lower (P < .05) in male rats fed diets containing 0 and 1 mg Cu/kg and female rats fed diet containing 0 mg Cu/kg compared with rats fed diet containing 6 mg Cu/kg. These changes were similar to changes in liver Cu concentrations, liver cyt c oxidase (CCO) activity and plasma ceruloplasmin in males and females. Serum SOD3 activity was also strongly, positively correlated with liver Cu concentrations over the entire range of dietary Cu concentrations (R(2) = .942 in males, R(2) = .884 in females, P < .0001). Plots of serum SOD3 activity, liver Cu concentration, liver CCO activity and ceruloplasmin as functions of kidney Cu concentration all had two linear segments that intersected at similar kidney Cu concentrations (18-22 microg/g dry kidney in males, 15-17 microg/g dry kidney in females). These findings indicate that serum SOD3 activity is a sensitive index of Cu status.     

Reactivity of antiinflammatory and superoxide dismutase active Cu(II)-salicylates.

The activity of chelated Cu(II) with four different aspirin-like drugs in various superoxide dismutase assays was examined. Prior to these studies the oxidation state of the involved copper was measured by x-ray photoelectron spectrometry and was found to be +II throughout. All copper complexes were able to suppress the xanthine-xanthine oxidase mediated reduction of both cytochrome c and nitroblue tetrazolium as well as the formazan formation by KO2 in a specific manner. The hydroxylation of benzo-[alpha]-pyrene as well as the demethylation of 7-ethoxycoumarin using induced hepatic rat microsomes could be successfully inhibited by the employed Cu(II) chelates. Cu(II)-acetylsalicylate was the most active copper complex. Our findings support the proposal that Cu(II) chelates are the active forms of aspirin-like antiinflammatory agents.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.     

Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases.     

Lipid peroxidation induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system.

Superoxide radicals in high concentrations were generated from alkaline H2O2 without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H2O2 concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H2O2 led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

Peroxidation of lysozyme treated with Cu(II) and hydrogen peroxide

When lysozyme was treated with Cu(II) and H₂O₂ at pH 7.4, the protein underwent polymerization as well as changes in its fluorescent characteristics. Upon prolonged incubation, most of the protein aggregates were degraded into smaller peptides. Amino acid analysis indicated that the basic amino acid residues were most susceptible to the oxidation. Tryptophan residues were converted to N-formylkynurenine and kynurenine, and lysine residues were deaminated to form α-aminoadipic acid δ-semi- aldehyde. During Cu(II)H₂O₂ treatment, the formation of carbonyl groups was accompanied by the loss of free amino groups in the protein. Succinylation of free amino groups protected lysine residues from oxidation by Cu(II)H₂O₂, but failed to prevent polymerization. The studies with the modified lysozyme suggest that Cu(II)H₂O₂ can oxidize various amino acid residues in addition to lysine to generate different types of carbonyl compounds and these carbonyl compounds may be responsible for the formation of crosslinks in the polymerization process.     

Simulation of the diffusion-controlled reaction between superoxide and superoxide dismutase. I. Simple models

A Brownian dynamics simulation method is used to study the diffusion-influenced bimolecular reaction between superoxide and superoxide dismutase (SOD). Using simple models, the details of which are based on the crystallographic structure of SOD, it is found that the electrostatic charge distribution of SOD serves to guide superoxide into the active site and enhance the diffusion-controlled rate constant by about 40%.