The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

The role of lysyl residues of phospholipases A2 in the formation of the catalytic complex

The aspartyl residue at position 49 in phospholipases A2 (PLA) has been viewed as a component of the catalytic apparatus because of its involvement in binding the essential cofactor, calcium. We recently discovered a new class of PLA's in which, among other changes in highly invariant residues, Asp-49 is replaced by a lysine (Maraganore et al. (1984) J. Biol. Chem. 259, 13839). These Lys-49 PLA's are also calcium-dependent, but, in contrast to the Asp-49 enzymes, they bind phospholipid strongly in the absence of calcium. Lys-49 PLA's are, therefore, ideal for studying structural and mechanistic aspects of these enzymes. Attempts to modify Lys-49 with the amino group-specific reagent, trinitrobenzenesulfonic acid (TNBS) led to the inactivation of the PLA, but reaction occurred not as expected at position 49, but at Lys-53. These findings lead us to propose a model, applicable to PLA's in general, in which cationic side chains at position 53 in these enzymes participate in phospholipid binding on the path to formation of the catalytic complex. This model serves to explain a number of unresolved observations in the current literature relating to enzyme-substrate interactions in the PLA's.     

The role of superoxide radicals in lactoperoxidase-catalysed H2O2-metabolism and in irreversible enzyme inactivation

Irreversible inactivation of lactoperoxidase in the presence of excess H2O2 has been investigated. Serial overlay absorption spectra of the Soret region show that the rate and total amount of enzyme inactivation depend on the proton concentration. Perhydroxyl or superoxide radicals (HO.2 or O-2) cannot be established as the inactivating species in this mechanism, but they influence the rate of reconversion of the intermediate lactoperoxidase-compound III back to the resting ferric form of the enzyme.     

Mechanism of H2O2 production in porcine thyroid cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H2O2-generating machinery.

Hydrogen peroxide (H2O2), which is required for thyroid hormone synthesis, has been believed to be produced at the apical cell surface of thyroid follicular cells. However, we recently found that plasma membrane from porcine thyroid exclusively generated superoxide anion (O2-) by employing a novel method for simultaneous determination of H2O2 and O2- with diacetyldeuterioheme-substituted horseradish peroxidase (diacetyl-HRP) as the trapping reagent [Nakamura, Y., Ohtaki, S., Makino, R., Tanaka, T., & Ishimura, Y. (1989) J. Biol. Chem. 264, 4759-4761]. The present study describes the mechanism of H2O2 production as analyzed by this new method. Incubation of cultured porcine follicular cells with ionomycin, a Ca-ionophore, caused an increase in oxygen uptake of about 80%. During enhanced respiration, the cells released H2O2 in an amount equivalent to the amount of oxygen consumed as judged by the formation of compound II of diacetyl-HRP, and H2O2 adduct of the peroxidase. No formation of compound III of the peroxidase, an O2- adduct, was detected during burst respiration. Thus, the intact cells exclusively released H2O2 to the outside of the cells. On the other hand, when the cell fragments from follicular cells were incubated with NADPH or NADH in the presence of Ca2+, the production of O2- was observed only during NADPH-dependent burst respiration, supporting our previous results that the plasma membrane exhibited NADPH-dependent O2(-)-generating activity. O2- production by the plasma membrane was further confirmed by analyses of the effects of superoxide dismutase (SOD) and catalase on the reaction. These results suggested that H2O2 is secondarily produced through the dismutation of O2-.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of H2O2 formation in the insulin-like and insulin antagonistic effects on fat cells of omega-aminoalkyl glycosides.

A class of ω-aminoalkyl glycosides previously found to antagonize insulin's action on glucose oxidation in fat cells and to stimulate glucose oxidation in insulin's absence is now shown to mimic insulin also on the conversion of glucose to free fatty acids and to glycerol and glycerides. These glycosides also act like insulin by inhibiting hormone- and cholera toxin-stimulated lipolysis. Various lines of evidence demonstrate that most, if not all, of the insulin-like activity of these glycosides results from H₂O₂ formed from an amine oxidase-catalyzed oxidation of the aminoalkyl moiety of these compounds. A contaminant in the bovine plasma albumin (BPA) preparations used in the bioassays was found to represent a major source of the amine oxidase activity. Membrane (ghost) preparations were also found to possess amine oxidase activity capable of forming H₂O₂ from the glycosides in amounts sufficient to express insulin-like activity. Preliminary experiments with intact adipocytes suggest that this activity is located on the cell surface. The BPA-associated activity corresponds to the known Cu²⁺-containing “plasma-type” amine oxidase (EC 1.4.3.6) on the basis of its substrate specificity and susceptibility to selective inhibitors. The plasma membrane activity appears to correspond to neither the plasma-type nor to the flavin-containing mitochondrial-type (EC 1.4.3.4) and remains to be identified. The observed potent antilipolytic effects of both H₂O₂ and the aminoalkyl glycosides points out that any mechanism used to explain the insulin-like action of H₂O₂ must account for this ability to inhibit lipolysis as well as to stimulate glucose utilization. That catalase inhibits the insulin-like action of the glycosides and H₂O₂, but not that of insulin indicates that insulin's action is not mediated by cell surface-produced H₂O₂. Also, since the insulin antagonistic activity of these glycosides was not inhibited by catalase, H₂O₂ formation is not responsible for this antagonism. The latter finding, added to present and previous evidence on the carbohydrate structural requirements involved in H₂O₂ production and in the insulin-like biological and binding properties of the aminoalkyl glycosides, is consistent with a role(s) for their carbohydrate moieties in both the insulin antagonistic and agonistic activities of these compounds.     

Involvement of superoxide in the catalytic cycle of diamine oxidase.

The reaction of pig kidney diamine oxidase (amine:oxygen oxidoreductase (deaminating) (pyridoxal-containing), EC 1.4.3.6) could be significantly inhibited by superoxide dismutase active copper chelates but not by native 2Cu,2Zn-superoxide dimutase (cuprein). The ligands alone as well as Cd2+, a heavy metal of similar toxicity to Cu2+, showed no inhibition whatsoever. This indicates that .O-2 participates in the catalytic cycle and is produced at a site scarcely accessible to such a large molecule as cuprein. A mechanism for the second, aerobic step of the diamine oxidase reaction is suggested.     

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.     

The role of superoxide radical in the autoxidation of cytochrome c.

The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c. Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c. This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli. It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c. Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.     

Oxygen inhibition of nitroreductase: electron transfer from nitro radical-anions to oxygen.

The inhibition of many nitroreductases by oxygen has been explained by Mason and Holtzman in terms of electron transfer to oxygen from the nitro radical-anions, which have been identified as the first intermediate in some reductase systems. We have used the pulse radiolysis technique to measure the bimolecular rate constants of this electron-transfer reaction for over 20 nitro compounds, including substituted 2- and 5-nitroimidazoles of interest as antiprotozoal drugs and radiosensitizers, nitrofurans in use as antibacterial agents, and substituted nitrobenzenes previously used as model substrates for nitroreductases. The logarithm of the rate constant for the reaction of the nitro radical-anion with oxygen is linearly related to the one-electron reduction potential of the nitro compound.     

The role of beta93 Cys in the inhibition of Hb S fiber formation

Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the beta93 Cys residue. In this study, the role of beta93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of beta93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the beta93 and alpha104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the beta93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the beta93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the alpha104 residue in addition to the beta93 residue significantly perturbs the alpha(1)beta(2) interface, while modification of only beta93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the alpha104 Cys residue and not the beta93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.     

The mitochondrial site of superoxide formation

Ubiquinone and cytochrome b566 have both been postulated to cause mitochondrial O2 formation by autoxidation of their reduced forms. The present investigation was made to evaluate capabilities of the two candidates to transfer electrons to molecular oxygen out of sequence of the normal pathway of respiration. The results show that electron transfer from ubisemiquinone to oxygen depends on the availability of protons. In agreement with this finding autoxidation of redox cycling ubiquinone could not be observed due to its location in an aprotic environment of the mitochondrial membrane. However, O2 release from mitochondria was found to be related to the existence of low potential cytochrome b566. The transfer of this b type cytochrome to more positive values caused a concomitant decrease and finally inhibition of univalent electron transfer to oxygen out of sequence. Our findings suggest a role of cytochrome b 566 in mitochondrial O2 formation. A contribution by ubiquinone is unlikely as long as protons are deprived from penetrating into the domain where ubiquinone is operating.     

The involvement of the bridging imidazolate in the catalytic mechanism of action of bovine superoxide dismutase.

The pulse-radiolysis method has been used to study the catalytic mechanism of O2 leads to dismutation by the Co(II)-substituted bovine erythrocuprein (superoxide dismutase, EC 1.15.1.1). Catalysis is accompanied by spectral changes that may be interpreted in terms of rapid protonation and deprotonation of the Cu-facing nitrogen atom of the imidazolate that bridges the Cu(II) and the Co(II) [or Zn(II)] in the oxidized enzyme. This rapid change permits the possibility that the imidazole is a proton donor in the catalytic reduction of O2 leads to.     

Kinetics of the superoxide reductase catalytic cycle

The steady state kinetics of a Desulfovibrio (D.) vulgaris superoxide reductase (SOR) turnover cycle, in which superoxide is catalytically reduced to hydrogen peroxide at a [Fe(His)4(Cys)] active site, are reported. A proximal electron donor, rubredoxin, was used to supply reducing equivalents from NADPH via ferredoxin: NADP+ oxidoreductase, and xanthine/xanthine oxidase was used to provide a calibrated flux of superoxide. SOR turnover in this system was well coupled, i.e. approximately 2O*2 reduced:NADPH oxidized over a 10-fold range of superoxide flux. The reduction of the ferric SOR active site by reduced rubredoxin was independently measured to have a second-order rate constant of approximately 1 x 10(6) m-1 s-1. Analysis of the kinetics showed that: (i) 1 microM SOR can convert a 10 microM/min superoxide flux to a steady state superoxide concentration of 10(-10) m, during which SOR turns over about once every 6 s, (ii) the diffusion-controlled reaction of reduced SOR with superoxide is the slowest process during turnover, and (iii) neither ligation nor deligation of the active site carboxylate of SOR limits the turnover rate. An intracellular SOR concentration on the order of 10 microM is estimated to be the minimum required for lowering superoxide to sublethal levels in aerobically growing SOD knockout mutants of Escherichia coli. SORs from Desulfovibrio gigas and Treponema pallidum showed similar turnover rates when substituted for the D. vulgaris SOR, whereas superoxide dismutases showed no SOR activity in our assay. These results provide quantitative support for previous suggestions that, in times of oxidative stress, SORs efficiently divert intracellular reducing equivalents to superoxide.     

The role of the superoxide anion as a toxic species in the erythrocyte.

The toxic action of the superoxide anion (O₂^?) toward the erythrocyte was investigated with O₂^? generated through the autooxidation of dihydroxyfumaric acid (DHF). A suspension of human red cells exposed to DHF undergoes a rapid breakdown of the cellular hemoglobin to methemoglobin and other green pigments. This hemoglobin breakdown is inhibited by superoxide dismutase (SOD) or catalase (CAT) and is accelerated by lactoperoxidase (LP) added externally to the red cell medium. Associated with the hemoglobin breakdown is a hypotonic hemolysis also inhibited by SOD or CAT and initially accelerated but later inhibited by LP. Conversion of the red cell hemoglobin to carbonmonoxyhemoglobin in an aerated medium results in no hemoglobin breakdown or hypotonic lysis in the presence of DHF, even though O₂^? can be demonstrated in the medium. Although no evidence for membrane sulfhydryl oxidation or lipid peroxidation can be demonstrated in red cells exposed to DHF, the membranes of these cells were found to retain a green pigment. The presence of this green pigment in red cell membranes was inhibited by SOD, CAT, or conversion of the cellular hemoglobin to carbonmonoxyhemoglobin, but was not inhibited by LP. These results have been interpreted as a peroxide-dependent formation of O₂^? by DHF, followed by attack of O₂^? on hemoglobin. The reaction of O₂^? with hemoglobin leads to the formation of a hemoglobin-breakdown product that binds to the red cell membrane, resulting in an increased osmotic fragility of the cell.     

The biological role of superoxide dismutase

Recent data available in literature on mechanisms for regulation of the activity of superoxide dismutase (an antioxidant enzyme) and its interrelation to other enzymes and antioxidants are generalized. The role of superoxide dismutase in the ontogenesis and under different pathologies accompanied by the formation of free radicals is considered.     

Adriamycin stimulated superoxide formation in submitochondrial particles.

Adriamycin (doxorubicin), an anticancer agent, stimulated the formation of superoxide in submitochondrial particles isolated from bovine heart. Superoxide formation was detected by oxygen uptake, by the cooxidation of epinephrine to adrenochrome and by the reduction of acetylated cytochrome c. These processes were sensitive to superoxide dismutase (SOD). Rotenone-insensitive oxidation of NADH by the mitochondrial respiratory chain in the presence of oxygen caused the formation of approx 4 nmol of superoxide per min/mg of protein. Adriamycin at a concentration of 400 micron stimulated the rate of superoxide formation 6-fold to 25 nmol.min-1.mg-1, but this was not a maximum rate. Approximately 50 micron adriamycin was estimated to be sufficient for obtaining one-half maximal stimulation. Hydrogen peroxide accumulated as a final reaction product. Measurements of the relative catalase activity of blood-free tissues of rabbits and rats indicated that heart contained 2 to 4% of the catalase activity of liver or kidney. An enhanced production of superoxide and hydrogen peroxide and the relatively low catalase content of heart tissue may be factors in the cardiotoxicity induced by adriamycin chemotherapy if a similar reaction occurs in vivo.