The relative importance of HO* and ONOO- in mediating the toxicity of O*-.
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Chemical Phenomena↗
Biomedical subjects
I Fridovich
Publications and source records attributed to I Fridovich.
On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase.
The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).
Benzothiazoles↗
Catalytic scavenging of peroxynitrite by isomeric Mn(III) N-methylpyridylporphyrins in the presence of reductants.
Three isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin (MnTMPyP) were evaluated for their reaction with peroxynitrite. The Mn(III) complexes reacted with peroxynitrite anion with rate constants of 1.85 x 10(7), 3.82 x 10(6), and 4.33 x 10(6) M(-1) s(-1) at 37 degrees C for MnTM-2-PyP, MnTM-3-PyP, and MnTM-4-PyP, respectively, to yield the corresponding oxo-Mn(IV) complexes. Throughout the pH range from 5 to 8.5, MnTM-2-PyP reacted 5-fold faster than the other two isomers. The oxo-Mn(IV) complexes could in turn be reduced by glutathione, ascorbate, urate, or oxidize tyrosine. The rate constants for the reduction of the oxo-Mn(IV) complexes ranged from >10(7) M(-1) s(-1) for ascorbate to 10(3)-10(4) M(-1) s(-1) for tyrosine and glutathione. Cyclic voltammetry experiments show that there is no significant difference in the E1/2 of the Mn(IV)/Mn(III) couple; thus, the differential reactivity of the three isomeric complexes is interpreted in terms of electrostatic and steric effects. Micromolar concentrations of MnTM-2-PyP compete well with millimolar CO2 at reacting with ONOO-, and it can even scavenge a fraction of the ONOOCO2- that is formed. By being rapidly oxidized by ONOO- and ONOOCO2- and reduced by antioxidants such as ascorbate, urate, and glutathione, these manganese porphyrins, and especially MnTM-2-PyP, can redirect the oxidative potential of peroxynitrite toward natural antioxidants, thus protecting more critical targets such as proteins and nucleic acids.
Ascorbic Acid↗
Superoxide and iron: partners in crime.
Superoxide (O2-) poses multiple threats, which are diminished by a family of metalloenzymes, the superoxide dismutases. Among the damaging effects of O2- are direct oxidation of low-molecular-weight reductants; inactivation of a select group of enzymes; and reaction with NO to yield the strong oxidant, peroxynitrite. Of even greater import is the ability of O2- to univalently oxidize the [4 Fe-4 S] clusters of dehydratases, which causes release of iron. The "free" iron, which is kept reduced by cellular reductants, then reduces hydroperoxides to hydroxyl or alkoxyl radicals. Because the "free" iron will preferentially bind to anionic polymers, such as nucleic acids, or to anionic surfaces, such as cell membranes, these radicals will be generated adjacent to these vital targets and will preferentially attack them. O2- and iron can thus be viewed as partners in crime, and reciprocal regulatory effects between iron and O2- may be anticipated. These are discussed.
Escherichia coli↗
Fundamental aspects of reactive oxygen species, or what's the matter with oxygen?
A byproduct of normal aerobic metabolism is the generation of dangerously reactive intermediates of the reduction of O2. These include O2-, H2O2, and HO. and arise because of the predisposition of O2 for univalent reductions. These reactive oxygen species (ROS), and others that they can engender, threaten all cellular macromolecules, and defenses are needed. Among those known to date are: superoxide dismutases to convert O2- into O2 + H2O2; catalases to dismute H2O2 into O2 + H2O; and peroxidases to reduce H2O2 into 2H2O) and to reduce ROOH into ROH and H2O. These defenses are aided by enzymes that repair or recycle oxidatively damaged nucleic acids and proteins. A role for such oxidative damage in aging and neurodegenerative diseases is well supported.
Aerobiosis↗
Lucigenin as mediator of superoxide production: revisited.
Lucigenin caused a concentration-dependent increase in superoxide production by xanthine oxidase plus xanthine. This was seen, in terms of superoxide dismutase-inhibitable reduction of cytochrome c; in spite of the ability of univalently reduced lucigenin to directly reduce cytochrome c. It follows that in the absence of this interference, by the cytochrome, an even greater increase in superoxide production mediated by lucigenin would have been observed. Clearly lucigenin luminescence should not be relied upon as a method for measurement of, or even for detection of, superoxide.
Acridines↗
Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical.
The fluorogenic oxidation of hydroethidine (HE) to ethidium (E+) has been used as a measure of O2-. Evaluation of this method confirms that O2-, but not O2 or H2O2, rapidly oxidizes HE to E+. However the ratio of E+ produced per O2- introduced decreased as the flux of O2- was increased. This suggested that HE can catalyze the dismutation of O2- and this was affirmed. HE was oxidized to a red product, distinct from E+ by ferricytochrome c and a similar oxidation may occur within Escherichia coli. HE inhibited the growth and killed a SOD-null strain to a greater extent than the SOD-replete parental strain and these effects were much diminished under anaerobic conditions. This indicated that E+ was responsible for the toxicity of HE and indeed E+ was seen to be toxic under both aerobic and anaerobic conditions. In view of the data presented HE can be recommended as a qualitative but not as a quantitative measure of O2(-1).
Acridines↗
Superoxide dependence of the toxicity of short chain sugars.
Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain. A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine. Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner. An unidentified dialyzable component in E. coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD. The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds. In keeping with this view was the O-2-independent toxicity of methylglyoxal.
Acetaldehyde↗
The ortho effect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin a powerful and potentially useful superoxide dismutase mimic.
The ortho, meta, and para isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-3-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (SOD) activity in vitro and in vivo. The impact of their interaction with DNA and RNA on the SOD activity in vivo and in vitro has also been analyzed. Differences in their behavior are due to the combined steric and electrostatic factors. In vitro catalytic activities are closely related to their redox potentials. The half-wave potentials (E1/2) are +0.220 mV, +0.052 mV, and +0.060 V versus normal hydrogen electrode, whereas the rates of dismutation (kcat) are 6.0 x 10(7), 4.1 x 10(6), and 3.8 x 10(6) M-1 s-1 for the ortho, meta, and para isomers, respectively. However, the in vitro activity is not a sufficient predictor of in vivo efficacy. The ortho and meta isomers, although of significantly different in vitro SOD activities, have fairly close in vivo SOD efficacy due to their similarly weak interactions with DNA. In contrast, due to a higher degree of interaction with DNA, the para isomer inhibited growth of SOD-deficient Escherichia coli.
Catalysis↗
Growth in iron-enriched medium partially compensates Escherichia coli for the lack of manganese and iron superoxide dismutase.
Enrichment of the growth medium with iron partially relieves the phenotypic deficits imposed on Escherichia coli by lack of both manganese and iron superoxide dismutases. Thus iron supplementation increased the aerobic growth rate, decreased the leakage of sulfite, and diminished sensitivity toward paraquat. Iron supplementation increased the activities of several [4Fe-4S]-containing dehydratases, and this was seen even in the presence of 50 microg/ml of rifampicin, an amount which completely inhibited growth. Assessing the O-2 scavenging activity by means of lucigenin luminescence indicated that the iron-enriched sodAsodB cells had gained some means of eliminating O-2, which was not detectable as superoxide dismutase activity in cell extracts. It is noteworthy that iron-enriched cells were not more sensitive toward the lethality of H2O2 despite having the usual amount of catalase activity. This indicates that iron taken into the cells from the medium is not available for Fenton chemistry, but is available for reconstitution of iron-sulfur clusters. We suppose that oxidation of the [4Fe-4S] clusters of dehydratases by O-2 and their subsequent reductive reconstitution provides a mechanism for scavenging O-2 and that speeding this reductive reconstitution by iron enrichment both spared other targets from O-2 attack and maintained adequate levels of these enzymes to meet the metabolic needs of the cells.
Culture Media↗
The familial amyotrophic lateral sclerosis-associated amino acid substitutions E100G, G93A, and G93R do not influence the rate of inactivation of copper- and zinc-containing superoxide dismutase by H2O2.
Inactivation of copper- and zinc-containing superoxide dismutase (Cu,ZnSOD) by H2O2 is the consequence of several sequential reactions: reduction of the active site Cu(II) to Cu(I) by H2O2; oxidation of the Cu(I) by a second H2O2, thus generating a powerful oxidant, which may be Cu(I)O or Cu(II)OH or Cu(III); and finally oxidation of one of the histidines in the ligand field, causing loss of SOD activity. Three familial amyotrophic lateral sclerosis (FALS)-associated mutant Cu,ZnSODs, i.e., E100G, G93A, and G93R, did not differ from the control enzyme in susceptibility to inactivation by H2O2. It thus appears that an increased peroxidase activity of the FALS-associated Cu,ZnSOD variants might not be a factor in the development of this disease. This leaves the loss of Zn, and the consequent increase in peroxidase activity, or in nitration activity, as a viable explanation (J. P. Crow et al., 1997, J. Neurochem. 69, 1936-1944), among other possibilities.
Amyotrophic Lateral Sclerosis↗
The trail to superoxide dismutase.
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Biochemistry↗
Oxygen toxicity: a radical explanation.
During its reduction to water, O2 readily gives rise to dangerously reactive intermediates. This threat is diminished by families of defensive enzymes which include the superoxide dismutases, catalases and peroxidases. Free radical chain reactions are controlled by antioxidants, such as ascorbate and the tocopherols, and oxidative damage, which occurs in spite of these defenses, is largely repaired or is nullified by de novo biosynthesis. Yet some damage is sustained and it contributes to mutagenesis, to senescence and to numerous pathological processes.
Animals↗
Adverse effects of superoxide and defenses: studies with Escherichia coli.
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Escherichia coli↗
Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury.
Manganic porphyrins are redox active metal complexes that have been employed as superoxide dismutase mimics. We tested whether these metalloporphyrins could also dismute hydrogen peroxide (H2O2) and whether they could protect endothelial cells against H2O2. Both of the manganic metalloporphyrins tested were found to catalytically dismute H2O2. These manganic porphyrins also protected endothelial cells in dose-dependent manners against H2O2-mediated injury with MnTMPyP having an EC50 of 8 microM and MnTBAP having an EC50 of 15 microM. The zinc containing analogs of these porphyrins were inactive in dismuting H2O2 and did not protect. These studies further define the antioxidant capacity of metalloporphyrins in converting superoxide to H2O2 and H2O2 to water. These data suggest that manganic porphyrins may be useful therapeutics against disease states associated with the overproduction of reactive oxygen species.
Animals↗
Superoxide imposes leakage of sulfite from Escherichia coli.
Escherichia coli, which lacks the cytosolic superoxide dismutases, exhibits several nutritional auxotrophies when growing aerobically. The cysteine/methionine requirement, which is one of these, was previously shown to be due to leakage from the cells, and accumulation in the medium, of a metabolic intermediate on the biosynthetic route to these amino acids. The parental strain does not significantly accumulate this compound. It is now shown that treatment with alkaline cyanide releases sulfite from this compound, a property shared by alpha-hydroxy sulfonic acids (carbonyl-bisulfite adducts). Since E. coli accumulates carbonyl compounds in the growth medium, it appears likely that the sulfitogenic compounds accumulated by the sodA sodB strain are alpha-hydroxy sulfonic acids.
Cysteine↗
Superoxide-dependent peroxidase activity of H48Q: a superoxide dismutase variant associated with familial amyotrophic lateral sclerosis.
Approximately 20% of cases of familial amyotrophic lateral sclerosis are caused by dominant mutations in the Cu,Zn superoxide dismutase. One such mutant, in which histidine #48 has been replaced by glutamine (H48Q), exhibits a novel activity. It can react sequentially with O2- and H2O2 to generate a potent oxidant at its active site, possibly Cu(II)-OH, which then can oxidize urate to the corresponding radical. This O2- -dependent peroxidase activity exerted on a substrate peculiar to motor neurons may be the toxic gain of function which leads to the deleterious consequences of this mutation. G93A, G93R, and E100G were also examined and found not to exert this O2- -dependent peroxidase activity.
Amyotrophic Lateral Sclerosis↗
A mechanism for complementation of the sodA sodB defect in Escherichia coli by overproduction of the rbo gene product (desulfoferrodoxin) from Desulfoarculus baarsii.
Overexpression of rbo in Escherichia coli prevents the inactivation of the [4Fe-4S]-containing fumarases that otherwise occurs in the sodA sodB strain. It similarly protects against the increased sensitivity toward H2O2, which is imposed by the lack of SOD A and SOD B. These results would be explained on the basis of scavenging of O-2 within the cells by RBO. This interpretation was supported by measurements of intracellular scavenging of O-2 by the lucigenin luminescence method. Since SOD activity could not be detected in dilute extracts, of the RBO-overexpressing sodA sodB strain, we propose that RBO catalyzes the reduction of O-2 at the expense of cellular reductants such as NAD(P)H. A similar mechanism may apply to other instances of complementation of SOD defects by non-SOD genes.
Acridines↗