Superoxide anion radical (O2-.), superoxide dismutases, and related matters.
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Animals↗
Biomedical subjects
I Fridovich
Publications and source records attributed to I Fridovich.
A potent superoxide dismutase mimic: manganese beta-octabromo-meso-tetrakis-(N-methylpyridinium-4-yl) porphyrin.
Variously modified metalloporphyrins offer a promising route to stable and active mimics of superoxide dismutase (SOD). Here we explore bromination on the pyrroles as a means of increasing the redox potentials and the catalytic activities of the copper and manganese complexes of a cationic porphyrin. Mn(II) and Cu(II) octabrominated 5,10,15,20-tetrakis-(N-methylpyridinium-4-yl) porphyrin, Mn(II)OBTMPyP4+, and Cu(II)OBTMPyP4+ were prepared and characterized. The rate constants for the porphyrin-catalyzed dismutation of O2.- as determined from the inhibition of the cytochrome c reduction are k(cat) = 2.2 x 10(8) and 2.9 x 10(6) M(-1) s(-1), i.e., IC50 was calculated to be 12 nM and 0.88 microM, respectively. The metal-centered half-wave potential was E(1/2) = +0.48 V vs NHE for the manganese compound. Cu(II)OBTMPyP4+ proved to be extremely stable, while its Mn(II) analog has a moderate stability, log K = 8.08. Nevertheless, slow manganese dissociation from Mn(II)OBTMPyP4+ enabled the complex to persist and exhibit catalytic activity even at the nanomolar concentration level and at biological pH. The corresponding Mn(III)OBTMPyP5+ complex exhibited significantly increased stability, i.e., demetallation was not detected in the presence of a 400-fold molar excess of EDTA at micromolar porphyrin concentration and at pH 7.8. The beta-substituted manganese porphyrin facilitated the growth of a SOD-deficient strain of Escherichia coli when present at 0.05 microM but was toxic at 1.0 microM. The synthetic approach used in the case of manganese and copper compounds offers numerous possibilities whereby the interplay of the type and of the number of beta substituents on the porphyrin ring would hopefully lead to porphyrin compounds of increased stability, catalytic activity, and decreased toxicity.
Catalysis↗
The copper- and zinc-containing superoxide dismutase from Escherichia coli: molecular weight and stability.
The periplasmic Cu,Zn superoxide dismutase (Cu,ZnSOD) from Escherichia coli has been shown by sedimentation equilibrium to be a monomer with a molecular weight of approximately 17,000. The enzyme suffered a reversible inactivation when heated to 70 degrees C. This was minimized by added Cu(II) or Zn(II). Heat lability was greater in phosphate than in Tris buffer. The enzyme exhibited a time-dependent inactivation by Hg(II) and this too was greater in phosphate than in Tris. This behavior can be explained by a modest affinity of the enzyme for Cu(II) and Zn(II) which results in a dissociation/association equilibrium. Elevation of the temperature shifts this equilibrium toward dissociation and phosphate sequesters the released metals making them less available for reinsertion at the active site. Hg(II) competes for occupancy of the active site and there were more unoccupied sites in phosphate than in Tris. A parallel was drawn between the E. coli Cu,ZnSOD and FALS varients of human Cu,ZnSOD, which are also relatively unstable and exhibit low affinity for Cu(II).
Azides↗
Lucigenin luminescence as a measure of intracellular superoxide dismutase activity in Escherichia coli.
Lucigenin and paraquat are similar in that each can be taken into Escherichia coli and can then mediate O2.- production by cycles of univalent reduction, to the corresponding monocation radical, followed by autoxidation. Thus, both compounds caused induction of enzymes that are regulated by the soxRS regulon. The lucigenin cation radical has the added property of reacting with O2.-, in a radical-radical addition, to yield an unstable dioxetane, whose decomposition yields light. Superoxide dismutases (SOD), by decreasing [O2.-], inhibit light production and to the same degree inhibit other O2.(-)-dependent reactions in the cell. Lucigenin luminescence was used to show that the levels of SOD in the parental strain provide approximately 95% protection of all O2.(-)-sensitive targets in E. coli. This degree of protection was so close to the limit of 100% that halving the parental level of [SOD], or increasing it 5-fold, had only marginal effects on the intensity of lucigenin-dependent luminescence.
Acridines↗
Lucigenin (bis-N-methylacridinium) as a mediator of superoxide anion production.
Lucigenin (bis-N-methylacridinium) (Luc2+) has frequently been used for the luminescent detection of O2-. In fact, the pathway leading to this luminescence requires univalent reduction of Luc2+ to LucH.+ followed by formation of an unstable dioxetane by reaction of LucH.+ with O2-. It is now shown that LucH.+ rapidly autooxidizes and so produces O2-. Luc2+ can thus mediate O2- production in systems, such as glucose plus glucose oxidase, in which there is ordinarily no O2- production. Luc2+ luminescence can thus be used as the basis for assaying superoxide dismutase activity but should not be used for measuring, or even detecting, O2-.
Acridines↗
How does superoxide dismutase protect against tumor necrosis factor: a hypothesis informed by effect of superoxide on "free" iron.
The manganese-containing mitochondrial superoxide dismutase (MnSOD) is induced by TNF and protects against the necrotic effect of this cytokine. Yet TNF does not increase production of O2- in mitochondria. How is this to be reconciled? TNF is known to increase production of arachidonate, by activation of phospholipase A2 (PLA2). Arachidonate will be converted to the corresponding alkyl hydroperoxide by lipoxygenase. O2- increases "free" iron by oxidizing [4Fe-4S] clusters of dehydratases, such as aconitase. Ferrous iron in turn reacts with alkyl hydroperoxides, in an analogue of the Fenton reaction, to produce alkoxyl radicals: which can initiate the oxidation of polyunsaturated lipids by a free radical chain reaction. MnSOD protects against TNF by decreasing O2- attack on [4Fe-4S] clusters and thus lowering free iron. Inhibitors of PLA2 and of lipoxygenase should also protect by decreasing fatty acyl hydroperoxides and they are known to do so. Cells having little mitochondrial MnSOD, or cells unable to induce that defensive enzyme in response to TNF, will consequently have relatively high levels of "free" iron in that organelle; leading to enhanced lipid peroxidation. Such cells will be preferentially killed by this cytokine.
Animals↗
The rate of adaptive mutagenesis in Escherichia coli is enhanced by oxygen (superoxide).
Adaptive mutagenesis is that which occurs in non-dividing cells and which allows growth under the selective conditions imposed. We now report that reversion of amino acid auxotrophies in E. coli fits that definition and is enhanced under conditions conducive to oxidative damage to DNA. Thus adaptive mutagenesis was approximately 4-fold more frequent in a sodA sodB strain than in the superoxide dismutase-replete parental strain and this mutagenesis was suppressed under anaerobic conditions. Moreover, a cell permeant manganic porphyrin, capable of catalyzing the dismutation of O2-, diminished the rate of occurrence of these mutations. Repair of oxidative damage to DNA, in the non-dividing cells, appears to provide the opportunity for adaptive mutagenesis.
Amidohydrolases↗
The mechanism of the auxotrophy for sulfur-containing amino acids imposed upon Escherichia coli by superoxide.
Defects in both of the genes coding for the cytosolic superoxide dismutases (SODs) of Escherichia coli impose an oxygen-dependent nutritional requirement for cysteine. This is now seen to be a bradytrophy, rather than an absolute auxotrophy, since lack of Cys merely imposed a growth lag and escape from this growth lag did not involve genetic reversion. This Cys bradytrophy was not seen in the SOD-competent parental strain, and it was relieved by a cell-permeant mimic of SOD activity; hence, it was due to O2-.. It was also relieved by an osmolyte, such as sucrose; hence, it appears due to leakage from the cell of some component needed for Cys biosynthesis. Medium conditioned by the aerobic growth of the SOD-defective strain relieved the growth lag. Bioassays with Cys mutants suggested that the conditioned medium contained SO3-3 or its equivalent, and sulfite per se was able to eliminate the growth lag. However, some component of the conditioned medium reacted with added sulfite and interfered with attempts to assay for it colorimetrically. These results suggest that the cell envelope of the SOD-defective strain was weakened, directly or indirectly, by O2 and then leaked sulfite. This prevents cysteine biosynthesis until sulfite accumulates in the medium.
Culture Media, Conditioned↗
Superoxide dismutase activity in the giant hemoglobin of the earthworm, Lumbricus terrestris.
The giant hemoglobin, which occurs in free solution in the blood of earthworms, contains copper and zinc and exhibits superoxide dismutase activity as assessed by competition assays using cytochrome c or nitroblue tetrazolium. On a molar basis, the activity of this hemoglobin was approximately 10% that of the mammalian copper, zinc superoxide dismutase. The dodecamer, which contains the globin chains but lacks the linker subunits, had very little activity when compared to the complete native molecule. This suggests that the superoxide dismutase activity resides in one of the linker subunits. This is the first report of a superoxide dismutase bound to a respiratory pigment.
Animals↗
Escherichia coli exhibits negative chemotaxis in gradients of hydrogen peroxide, hypochlorite, and N-chlorotaurine: products of the respiratory burst of phagocytic cells.
Escherichia coli can respond to gradients of specific compounds, moving up gradients of attractants and down gradients of repellents. Stimulated phagocytic leukocytes produce H2O2, OCl-, and N-chlorotaurine in a response termed the respiratory burst. E. coli is actively repelled by these compounds. Catalase in the suspending medium eliminated the effect of H2O2. Repulsion by H2O2 could be demonstrated with 1 microM H2O2, which is far below the level that caused overt toxicity. Strains with defects in the biosynthesis of glutathione or lacking hydroperoxidases I and II retained this response to H2O2, and 2.0 mM CN- did not interfere with it. Mutants with defects in any one of the four known methyl-accepting chemotaxis proteins also retained the ability to respond to H2O2, but a "gutted" mutant that was deleted for all four methyl-accepting chemotaxis proteins, as well as for CheA, CheW, CheR, CheB, CheY, and CheZ, did not respond to H2O2. Hypochlorite and N-chlorotaurine were also strongly repellent. Chemotaxis down gradients of H2O2, OCl-, and N-chlorotaurine may contribute to the survival of commensal or pathogenic microorganisms.
Animals↗
Functional significance of the Cu,ZnSOD in Escherichia coli.
Diethyl dithiocarbamate (DDC) was used to inhibit the copper- and zinc- containing superoxide dismutase (Cu,ZnSOD) of Escherichia coli in order to expose its physiological function. DDC inhibited the aerobic growth of a sodA sodB mutant much more than it did the growth of a SOD-replete parental strain and this inhibitory effect was oxygen-dependent. The SOD mimic MnTMPyP markedly diminished the growth inhibitory effect of DDC. Transfer of the sodA sodB strain from anaerobic to aerobic conditions induced fumarase C, which is a member of the soxRS regulon. DDC augmented this induction. These results indicate that the Cu,ZnSOD provides a defense against oxidative stress, which is more important to the sodA sodB mutant than to its SOD-replete parental strain.
Aerobiosis↗
Comments on vanadate chemistry.
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Electron Spin Resonance Spectroscopy↗
Purification and characterization of the Cu,Zn SOD from Escherichia coli.
The periplasmic Cu,Zn superoxide dismutase has been purified to homogeneity by a procedure, which depended upon osmotic shock followed by two chromatographic columns. Its subunit weight, determined by electrospray ionization mass spectrometry, was found to be 15,737 +/- 1.6. The second derivative ultraviolet spectrum indicated a lack of tryptophan. The amino acid composition as well as a partial N-terminal amino acid sequence is reported. The specific activity was 3700 U/mg and the corresponding copper content was 0.77 atoms Cu/subunit. The enzyme was quite unstable and overnight dialysis against EDTA or even prolonged dialysis against neutral phosphate buffer caused partial loss of activity and of copper and visible precipitation. It is likely that some losses occurred during the isolation procedure, and if these could have been prevented the copper content would have been 1.0 Cu/subunit and the specific activity would have been 4800 U/mg. It now appears likely that gram negative bacteria will commonly be found to contain a periplasmic Cu,Zn SOD.
Amino Acid Sequence↗
Measuring nitric oxide and superoxide: rate constants for aconitase reactivity.
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Aconitate Hydratase↗
Requirement for superoxide in excitotoxic cell death.
We tested the pathogenic role of O2-) radicals in excitotoxic injury. Inactivation of the TCA cycle enzyme, aconitase, was used as a marker of intracellular O2- levels, and a porphyrin SOD mimetic was used to scavenge O2-. The selective, reversible, and SOD-sensitive inactivation of aconitase by known O2- generators was used to validate aconitase activity as a marker of O2- generation. Treatment of rat cortical cultures with NMDA, KA, or the intracellular O2- generator PQ2+ produced a selective and reversible inactivation of aconitase, which closely correlated with subsequent cell death produced by these agents. The SOD mimetic, but not its less active congener, attenuated both aconitase inactivation and cell death produced by NMDA, KA, and PQ2+. These results provide direct evidence implicating O2(-) generation in the pathway to excitotoxic injury.
Aconitate Hydratase↗
Red blood cell antioxidant enzymes in age-related macular degeneration.
AIMS/BACKGROUND: Oxidative damage has been proposed to be involved in the pathogenesis of age-related macular degeneration (ARMD). The purpose of this study was to evaluate whether red blood cell antioxidant enzyme activity correlates with severity of aging maculopathy in affected individuals. METHODS: Blood samples were obtained from 54 patients with varying severity of aging maculopathy and 12 similarly aged individuals with normal ophthalmoscopic examination. Macular findings were graded according to a modification of the method described for the Age-Related Eye Disease Study. (AREDS). The activities of superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, and glutathione reductase were measured in red blood cells. Haemoglobin content of whole blood was measured, and enzyme activity was determined per mg haemoglobin. RESULTS: Multiple regression analysis and ordinal logistic regression analysis were performed to determine whether antioxidant enzyme activity was associated with severity of ARMD. No significant association between disease severity of ARMD and antioxidant enzyme activity was identified for any of the enzymes. CONCLUSION: These results do not provide evidence for a relation between oxidative stress, as measured by antioxidant enzyme activity in red blood cells, and disease severity in ARMD.
Aged↗
Negative chemotaxis in Cytophaga johnsonae.
Chemotaxis, both positive and negative, has been extensively studied in flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, but not in gliding bacteria. The rapidly motile gliding bacterium Cytophaga johnsonae has been seen to be repelled by H2O2, OCl-, and N-chlorotaurine, as well as by low pH. Its response to H2O2 was eliminated by catalase. Nalidixic acid at 200 microM, which inhibits the growth but not the motility of C. johnsonae, did not interfere with its negative chemotactic response to H2O2, whereas sodium phosphate at 10 mM, which inhibits motility, did so. Cytophaga johnsonae was not repelled by taurine, n-octanol, phenol, L-valine, or high pH. Chemotaxis can be conveniently studied in gliding bacteria such as C. johnsonae.
Chemotaxis↗
Antioxidant enzymes of the human retina: effect of age on enzyme activity of macula and periphery.
The purpose of this research was to evaluate the effect of age on protective antioxidant enzyme activity of normal fresh cadaver human retina of the macula and periphery. Antioxidant enzymes were assayed in tissue extracts generated from 5 mm trephined punches of retina obtained centered over the macula and the superior midperiphery of normal fresh human cadaver retina. Cadaver tissue was obtained from donors of a wide age range (age 7 to 85 years). The assays were performed within 6 h of enucleation and within 24 h of donor death. Antioxidant enzymes assayed included superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Hexokinase and glucose-6-phosphate dehydrogenase, enzymes not directly involved in protection against oxidative damage, were assayed for comparison. Enzyme specific activities were calculated for the macula and periphery using protein concentration of the extract as the denominator. Using linear regression analysis, over the age range of 25 to 75 years, superoxide dismutase activity of the periphery but not the macula tended to decline with age (p = 0.04, R2 = 0.21). Interindividual variability was high, and variability increased with age. The difference between the macular and peripheral enzyme activities for glutathione peroxidase tended to decline with increasing donor age (p = 0.025, R2 = 0.33). There was no effect of age on the specific activities of catalase, glucose-6-phosphate dehydrogenase, and glutathione reductase. The specific activity of hexokinase from the macula declined with increasing donor age (p = 0.022, R2 = 0.43). Time from death to enucleation or beginning of experiment was not a significant factor. In summary, age does not have an effect on the activity of major antioxidant enzymes of the macula in normal human retina. There is a tendency for an effect of age on peripheral superoxide dismutase activity and the difference between macular and peripheral glutathione peroxidase activity. High interindividual variability of antioxidant enzyme activity exists in humans.
Adolescent↗