2-methoxyestradiol does not inhibit superoxide dismutase.

It has been reported in the literature that the endogenous estrogen metabolite 2-methoxyestradiol (2-ME) inhibits both manganese and copper,zinc superoxide dismutases (Mn and Cu,Zn SODs) and that this mechanism is responsible for 2-ME's ability to kill cancer cells. In fact, as demonstrated using several SOD assays including pulse radiolysis, 2-ME does not inhibit SOD but rather interferes with the SOD assay originally used. Nevertheless, as confirmed by aconitase inactivation measurements and lactate dehydrogenase release in human leukemia HL-60 cells, 2-ME does increase superoxide production in these cells and is more toxic than its non-O-methylated precursor 2-hydroxyestradiol. Other mechanisms previously suggested in the literature may explain 2-ME's ability to increase intracellular superoxide levels in tumor cells.

Copper,zinc superoxide dismutase as a univalent NO(-) oxidoreductase and as a dichlorofluorescin peroxidase.

Nitroxyl (NO(-)) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO(-) to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO(-) and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O(2) was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were approximately 100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO(-) caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O(2), SOD could catalyze the oxidation of NO(-) to NO. The reverse reaction, i.e. the reduction of NO to NO(-) by Cu(I),Zn-SOD, followed by the reaction of NO(-) with O(2) would yield ONOO(-) and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H(2)O(2) caused the HCO(3)(-)-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H(2)O(2) production.

The oxidation of 3-hydroxyanthranilic acid by Cu,Zn superoxide dismutase: mechanism and possible consequences.

Cu,Zn SOD, but not Mn SOD, catalyzes the oxidation of 3-hydroxyanthranilic acid (3-HA) under aerobic conditions. In the absence of O2, the Cu(II) of the enzyme is reduced by 3-HA. One plausible mechanism involves the reduction of the active site Cu(II) to Cu(I), which is then reoxidized by the O2- generated by autoxidation of the anthranilyl or other radicals on the pathway to cinnabarinate. We may call this the superoxide reductase, or SOR, mechanism. Another possibility invokes direct reoxidation of the active site Cu(I) by the anthranilyl, or other organic radicals, or by the peroxyl radicals generated by addition of O2 to these organic radicals. Such oxidations catalyzed by Cu,Zn SOD could account for the deleterious effects of the mutant Cu,Zn SODs associated with familial amyotrophic lateral sclerosis and of the overproduction or overadministration of wild-type Cu,Zn SOD.

Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase.

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O(2) as well as the reduction of ferricyanide by O(2). Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H(2)O(2) production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.

Lucigenin: redox potential in aqueous media and redox cycling with O-(2) production.

The use of lucigenin luminescence as a measure of ¿O-(2) has been questioned because lucigenin has been shown to be capable of mediating the production of O-(2). This being the case, lucigenin can signal the presence of O-(2) even in systems not producing it in the absence of lucigenin. The reduction potential of lucigenin should be in accord with its ability to mediate O-(2) production; but it has not heretofore been measured in aqueous media. The problems facing such measurement are the insolubility of the divalently reduced form, which deposits on the electrode, and the slow conformational transition that follows the second electron transfer and which interferes with reversibility. We have now used rapid scan cyclic voltammetry to determine that the reduction potential for lucigenin is -0.14 +/- 0.02 V versus the normal hydrogen electrode. This value applies to both the first and the second electron transfers to lucigenin and it is in accord with the facile mediation of O-(2) production by this compound.

Induction of the soxRS regulon of Escherichia coli by superoxide.

The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of approximately 15 genes. The induction of this regulon by redox agents, known to mediate O-2 production, led to the view that O-2 is one signal to which it responds. However, redox cycling agents deplete cellular reductants while producing O-2, and one may question whether the regulon responds to the depletion of some cytoplasmic reductant or to O-2, or both. We demonstrate that raising [O-2] by mutational deletion of superoxide dismutases and/or by addition of paraquat, both under aerobic conditions, causes induction of a member of the soxRS regulon and that a mutational defect in soxRS eliminates that induction. This establishes that O-2, directly or indirectly, can cause induction of this defensive regulon.

Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli.

Nitroreductase A catalyzes the divalent reduction of nitro compounds, quinones, and dyes by NADPH. In this paper, nitroreductase A is induced in Escherichia coli by exposure to paraquat in a manner that depends on the expression of soxR. Nitroreductase activity was only slightly induced by paraquat in a strain bearing a mutational defect in the gene encoding nitroreductase A, but it was approximately 3-fold induced in the parental strain. Nitroreductase A thus appears to be a member of the soxRS regulon and probably contributes to the defenses against oxidative stress by minimizing the redox cycling attendant upon the univalent reduction of nitro compounds, quinones, and dyes.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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-.

The role of iron-sulfur clusters in in vivo hydroxyl radical production.

The in vivo production of HO- requires iron ions, H2O2 and O2- or other oxidants but probably does not occur through the Haber-Weiss reaction. Instead oxidants, such as O2-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly. Fe(II) then reduces H2O2, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione. In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO. production to a minimum. O2- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein. The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction. In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the "free" iron concentration intracellularly both by decreasing the biosynthesis of ferritin and increasing biosynthesis of transferrin receptors. The biological role of the soxRS regulon of Escherichia coli, which is involved in the adaptation toward oxidative stress, is presumably to counteract the oxidative inactivation of the iron clusters and the subsequent release of iron with consequent increased rate of production of HO.