Reprint of: Biological Effects of the Superoxide Radical.

Can the superoxide radical exert deleterious effects independent of participating with H2O2 in the production of the hydroxyl radical? Examination of the superoxide-related literature reveals data suggesting an affirmative answer to this question. © 1986 Academic Press, Inc.

Reprint of: Intracellular Production of Superoxide Radical and of Hydrogen Peroxide by Redox Active Compounds.

Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of O2- and H2O2. One indication of this electron-shunting action was increased cyanide-resistant respiration and one cellular response was increased biosynthesis of the manganese-containing superoxide dismutase and of catalase. Blocking cytochrome oxidase with cyanide or azide increased the electron flow available for reduction of paraquat and presumably of the other exogenous compounds tested and thus increased their biological effects. Paraquat, pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, plumbagin, methylene blue, and azure C were all effective in elevating intracellular production of O2- and H2O2. The effect of alloxan appeared paradoxical in that it increased cyanide-resistant respiration without significantly increasing the cell content of the manganese-superoxide dismutase and with only a small effect on the level of catalase. The alloxan effect on cyanide-resistant respiration was artifactual and was due to an oxygen-consuming reaction between alloxan and cyanide, rather than to a diversion of the intracellular electron flow. With paraquat as a representative electron-shunting compound, the increase in biosynthesis of the manganese-superoxide dismutase was prevented by inhibitors of transcription or of translation, but not by an inhibitor of replication. The increase in this enzyme activity, caused by paraquat and presumably by the other compounds, was thus due to de novo enzyme synthesis activated or derepressed at the level of transcription.

Oxygen: how do we stand it?.

The electronic structure of ground state oxygen, which is essential for the life of all aerobic organisms, makes it potentially dangerous for those organisms. Atmospheric oxygen contains two unpaired electrons with parallel spin states, which predisposes it to reduction by a univalent pathway. As a consequence, normal aerobic metabolism generates dangerous reactive intermediates of the reduction of O2. These include superoxide radical (O2*-), hydrogen peroxide (H2O2), and hydroxyl radical (HO*). These reactive oxygen species and others that they can engender can damage all cellular macromolecules and unless opposed by cellular defenses, would make aerobic life impossible. Such defenses include superoxide dismutases, catalases, and peroxidases, enzymes that decrease the concentration of the reactive oxygen species that are their substrates, and others that repair or recycle oxidatively damaged macromolecules. Any factor that stimulates reactive oxygen species production or suppresses the antioxidant systems would inevitably cause cell damage. The role of such oxidative damage in various diseases is well documented. In vivo detection of O2- and other reactive oxygen species is however hampered by the lack of easy, specific, and sensitive analytical methods. Potential artifacts and limitations of the most common detection methods currently in use are briefly discussed.

Putative denitrosylase activity of Cu,Zn-superoxide dismutase.

The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations.

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.

Modification of cysteine 111 in human Cu,Zn-superoxide dismutase.

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5'-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.

Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content--order restored.

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234-240; 2005) the authors argued that the catalytic rate constant, k(cat), for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sample that already contained excess copper did not remove spurious activity, which is incompatible with well-known stability constants of copper complexes and contradicts previous observations. In the present study we verified that the addition of EDTA eliminates completely the effect of excess copper on the decomposition rate of O2*- in the presence of Cu,Zn-SOD. We determined that k(cat) = (2.82 +/- 0.02) x 10(9) M(-1) s(-1) at low ionic strength (2 < I < 15 mM) and (1.30 +/- 0.02) x 10(9) M(-1) s(-1) in the presence of 50 mM phosphate at pH 7.8 (I = approximately 150 mM), which are about twice higher than those reported by Michel et al. We also determined k(cat) by the cytochrome c assay and demonstrated the correlation between these direct and indirect assays. The phenotypic deficits imposed by deletion of SODs, and the oxygen dependence of these deficits, have repeatedly demonstrated that the several SODs do in fact, as well as is theory, provide an important protection against that facet of oxidative stress imposed by O2*-.

Inactivation of copper, zinc superoxide dismutase by H2O2 : mechanism of protection.

Cu,Zn SOD is known to be inactivated by HO(2)(-) and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO(2)(-). We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0-9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.

The role of CO2 in metal-catalyzed peroxidations.

Transition metals, such as Cu(+2), Mn(+2), and Co(+2), have been seen to catalyze the bicarbonate enhanced oxidation of a variety of substrates by H(2)O(2). In several of these cases it has been demonstrated that CO(2), rather than bicarbonate, is the enhancing species. Mechanisms that are in accord with the data involve a hypervalent state that may be written (MO)(+n), or (MOH)(+n+1), or (M)(+n+2). This metal centered oxidant then oxidizes CO(2) to the carbonate radical; that is then the proximal oxidant of the various substrates. Whether a similar process has in vivo reality remains to be demonstrated.

Cross-compartment protection by SOD1.

The absence of SOD1 in yeast has been found to result in inactivation of Lys4p. This [4Fe-4S]-containing dehydratase is in the pathway of biosynthesis of lysine, hence the oxygen-dependent lysine auxotrophy seen in this case. O(2)(-) is known to oxidize and thus destabilize the [Fe-4S] clusters of dehydratases; hence, this would make perfect sense were it not for the fact that SOD1 localizes to the cytosol and the intermembrane space of mitochondria, whereas Lys4p localizes to the mitochondrial matrix. How could SOD1 in one compartment protect against O(2)(-) attack in a different compartment? We suggest that the relatively high levels of O(2)(-) in the cytosol and intermembrane space of the SOD1 mutant may react with endogenous NO, forming HOONO that can diffuse into the mitochondrial matrix and there inactivate Lys4p and other [4Fe-4S]-containing dehydratases.

Mitochondria: are they the seat of senescence?

The frequently quoted figure for the fractional univalent reduction of oxygen to superoxide in mitochondria is certainly too high by at least one order of magnitude. This is so because the higher number (2%) was derived from mitochondria whose cytochrome c oxidase was blocked with cyanide. Nevertheless, even the more correct number (0.1%) means that the production of O(2)(-) and H(2)O(2) in mitochondria is large and apt to result in damage to macromolecules in spite of such defensive enzymes as superoxide dismutases and glutathione peroxidase. The data available for nematodes and flies provide a compelling case for the view that the accumulation of oxidative damage to specific mitochondrial proteins leads to the progressive dysfunction that we see as senescence. The data available from work with mammals are much weaker and do not yet allow a strong position to be taken.

Reversal of the superoxide dismutase reaction revisited.

Reversal of the superoxide dismutase (SOD) reaction was measured in terms of the reduction of tetranitromethane (TNM) by O2-. Cu,ZnSOD caused a biphasic reduction of TNM by H2O2. The rapid initial phase was stoichiometric with the enzyme and was followed by a slower catalytic phase that was oxygen dependent and was augmented by HCO3-. The reaction scheme explaining this behavior is presented and a rate constant for the reduction of O2 by the cuprous enzyme is estimated. This rate constant is so low that it precludes significant O2- production by the reduced enzyme under the conditions explored.

Mutant Cu,Zn superoxide dismutases and familial amyotrophic lateral sclerosis: evaluation of oxidative hypotheses.

FALS-associated missense mutations of SOD1 exhibit a toxic gain of function that leads to the death of motor neurons. The explanations for this toxicity fall into two broad categories. One involves a gain of some oxidative activity, while the second involves a gain of protein: protein interactions. Among the postulated oxidative activities are the following: (i) peroxidase action; (ii) superoxide reductase action; and, (iii) the enhancement of production of O2- by partial reversal of the normal SOD activity, which then leads to increased formation of ONOO(-). We will herein concentrate on evaluating the relative merits of these oxidative hypotheses and consider whether the experiments with transgenic animals that purport to disprove these oxidative explanations really do so.

The mode of decomposition of Angeli's salt (Na2N2O3) and the effects thereon of oxygen, nitrite, superoxide dismutase, and glutathione.

The classical view of the aerobic decomposition of Angeli's salt is that it releases NO(2)(-) + NO(-)/HNO the latter then reacting with O(2) to yield ONOO(-). An alternative that has recently been proposed envisions electron transfer to O(2) followed by decomposition to NO(2)(-) + NO. The classical view is now strongly supported by the observation that the rates of decomposition of Angeli's salt under 20% O(2) or 100% O(2) were equal. Moreover, NO(2)(-), which inhibits this decomposition by favoring the back reaction, was more effective in the absence of agents that scavenge NO(-)/HNO. It is thus clear that Angeli's salt is a useful source of NO(-)/HNO for use in defined aqueous systems. The measurements made in the course of this work allowed approximation of the rate constants for the reactions of NO(-)/HNO with NO(2)(-), O(2), glutathione, or Cu, Zn superoxide dismutase. The likelihood of the formation of NO(-)/HNO in vivo is also discussed.

CO2 enhanced peroxidase activity of SOD1: the effects of pH.

At pH 7.4, CO2, rather than HCO3-, markedly enhances the oxidation of diverse substrates by SOD1 plus H2O2. Since the concentration of CO2 would fall with rising pH in HCO3- buffers, it was of interest to explore the effects of pH on the peroxidase activity of SOD1 in the presence and in the absence of HCO3-. The rate of NADPH peroxidation in the HCO3- buffer was minimally affected by pH in the range of 8-10.5; in a pyrophosphate buffer, the rate increased markedly, such that at pH 10.5 the rates in the two buffers were nearly identical. Similar results were obtained when urate was used as the peroxidizeable substrate. These results are explicable on the basis of an increase in the rate with pH due to the ionization of H2O2 to the effective HO2- coupled with a decrease in [CO2] due to the ionizations of H2CO3, which displaces the hydration equilibrium to the right. These two opposing effects counteract in the HCO3(-)-buffered reaction mixtures; in the pyrophosphate buffer, only the effect of increasing [H02-] was seen.

Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin.

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. We now show that tetrahydrobiopterin (BH(4)) is also able to reduce the metal center. Under anaerobic conditions, in phosphate-buffered saline (pH 7.4) at 25 +/- 0.1 degrees C, reduction of Mn(III)TE-2-PyP(5+) occurs through two reaction steps with rate constants k(1) = 1.0 x 10(4) M(-1) s(-1) and k(2) = 1.5 x 10(3) M(-1) s(-1). We ascribe these steps to the formation of tetrahydrobiopterin radical (BH(4)(.+)) (k(1)) that then undergoes oxidation to 6,7-dihydro-8H-biopterin (k(2)), which upon rearrangement gives rise to 7,8-dihydrobiopterin (7,8-BH(2)). Under aerobic conditions, Mn(III)TE-2-PyP(5+) catalytically oxidizes BH(4). This is also true for its longer chain alkyl analog, Mn(III) ortho-tetrakis(N-n-octylpyridinium-2-yl)porphyrin. The reduced Mn(II) porphyrin cannot be oxidized by 7,8-BH(2) or by l-sepiapterin. The data are discussed with regard to the possible impact of the interaction of Mn(III)TE-2-PyP(5+) with BH(4) on endothelial cell proliferation and hence on tumor antiangiogenesis via inhibition of nitric oxide synthase.

Complementation of SOD-deficient Escherichia coli by manganese porphyrin mimics of superoxide dismutase activity.

Cationic Mn(III) porphyrins substituted on the methine bridge carbons (meso positions) with N-alkylpyridinium or N,N'-diethylimidazolium groups have been prepared and characterized, both chemically and as SOD mimics. The ortho tetrakis N-methylpyridinium compound was substantially more active than the corresponding para isomer. This ortho compound also exhibited a more positive redox potential and greater ability to facilitate the aerobic growth of a SOD-deficient Escherichia coli. Analogs with longer alkyl side chains and with methoxyethyl side chains, as well as with N,N'-diethylimidazolium and N,N'-dimethoxyethylimidazolium groups on the meso positions, have been prepared in anticipation of greater penetration of the cells due to greater lipophilicity. We now report that the more lipophilic compounds were effective at complementing the SOD-deficient E. coli at lower concentrations than were needed with the less lipophilic compounds. The greater efficacy of the more lipophilic compounds was achieved at the cost of greater toxicity that became apparent when these compounds were applied at higher concentrations.

A small molecular weight catalytic metalloporphyrin antioxidant with superoxide dismutase (SOD) mimetic properties protects lungs from radiation-induced injury.

Radiation therapy (RT) is an important therapeutic modality in the treatment of thoracic tumors. The maximum doses to these tumors are often limited by the radiation tolerance of lung tissues. Lung injury from ionizing radiation is believed to be a consequence of oxidative stress and a cascade of cytokine activity. Superoxide dismutase (SOD) is a key enzyme in cellular defenses against oxidative damage. The objective of this study was to determine whether the SOD mimetic AEOL 10113 [manganese (III) mesotetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP(5+))] increases the tolerance of lung to ionizing radiation. AEOL 10113 was able to significantly reduce the severity of RT-induced lung injury. This was strongly supported with histopathology results and measurements of collagen deposition (hydroxyproline content). There was a significant reduction in the plasma level of the profibrogenic cytokine transforming growth factor-beta (TGF-beta) in the group of rats receiving RT + AEOL 10113. In conclusion, the novel SOD mimetic, AEOL 10113, demonstrates a significant protective effect from radiation-induced lung injury.

The Haber-Weiss cycle -- 70 years later: an alternative view.

In a recent review published in this journal,(1) Koppenol traced the history of the Fenton reaction and of the catalytic decomposition of H(2)O(2) by iron salts. If his purpose was to shed light on current understanding of related chemistry in biological systems, he failed. Moreover, he managed to sow confusion by inaccurate reporting of the work of others. What follows is an attempt to point out these shortcomings and thus to clarify the situation.