Stefan I. Liochev

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 O are direct oxidation of low‐molecular2 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 O to univalently oxidize the [4 Fe2 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. O and iron can thus be viewed as part2 ners in crime, and reciprocal regulatory effects between iron and O2 may be anticipated. These are discussed.

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

Abstract In a recent review published in this journal, Koppenol traced the history of the Fenton reaction and of the catalytic decomposition of H2O2 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.

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.

Aluminum(III) facilitates the oxidation of NADH by the superoxide anion

Al(III) augments the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by enzymatic or photochemical sources of O2-. Superoxide dismutase, but not catalase, inhibited this action of Al(III). It thus appears that Al(III) forms a complex with O2-, which is a stronger oxidant than is O2- itself and which may contribute to the adverse biological effects of Al(III).

Mechanism of the peroxidase activity of Cu, Zn superoxide dismutase

In addition to its very efficient catalysis of the dismutation of superoxide ( O(2)(-) ) into O(2) plus H(2)O(2), Cu, Zn SOD acts less efficiently as a non-specific peroxidase. This peroxidase activity is CO(2) dependent although very slow peroxidation of some substrates occurs in the absence of CO2. The mechanism of that CO(2) dependence is explained by the generation of a strong oxidant at the copper site by two sequential reactions with H(2)O(2), followed by the oxidation of CO(2) to the carbonate radical that then diffuses into the bulk solution. This diffusible carbonate radical is then responsible for the diverse oxidations that have been reported. A different mechanism that involves the reduction of peroxymonocarbonate by the reduced superoxide dismutase to yield carbonate radical has been proposed. We will demonstrate that this mechanism is not supported by the available data. It seems likely that generation of the carbonate radical has relevance to the oxidative stress faced by aerobic organisms.

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.

Copper, Zinc Superoxide Dismutase and H2O2 EFFECTS OF BICARBONATE ON INACTIVATION AND OXIDATIONS OF NADPH AND URATE, AND ON CONSUMPTION of H2O2

Copper,zinc superoxide dismutase (Cu,Zn-SOD) catalyzes the HCO 3 − -dependent oxidation of diverse substrates. The mechanism of these oxidations involves the generation of a strong oxidant, derived from H2O2, at the active site copper. This bound oxidant then oxidizes HCO 3 − to a strong and diffusible oxidant, presumably the carbonate anion radical that leaves the active site and then oxidizes the diverse substrates. Cu,Zn-SOD is also subject to inactivation by H2O2. It is now demonstrated that the rates of HCO 3 − -dependent oxidations of NADPH and urate exceed the rate of inactivation of the enzyme by ∼100-fold. Cu,Zn-SOD is also seen to catalyze a HCO 3 − -dependent consumption of the H2O2 and that HCO 3 − does not protect Cu,Zn-SOD against inactivation by H2O2. A scheme of reactions is offered in explanation of these observations.

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 Angelis 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 Angelis 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 Angelis 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.

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 Angelis 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 ∼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 O2 would yield ONOO− and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H2O2 caused the HCO 3 − -dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H2O2 production.

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 ∼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.