472 Session 1: Superoxide and Superoxide Dismutases

I:1 SUPEROXIDE AND SUPEROXIDE DISMUTASES Irwin Fridovich Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 USA.

Oz-, which is easily made both in vitro and fi vlyo can act as either an oxidant or a reductant. The former action is favored by association with cationic centers such as Ht,

2Y) or Mn(II1). 0~~ rapidly oxidizes [4Fe-

clusters of dehydratases, such as aconitase, resulting in release of Fe(II); which can then reduce Hz02 to HO- t HO,. Viologens, quinones, benzofurazans and other compounds can divert electron flow from normal pathways and increase 0~~ production. 0 _ so made can be damaging, and SOD minimizes f hat damage. However diversion of electron flow is also damaging and SOD cannot influence that. For example the E. coli sulfite reductase readily transfers electrons to paraquat. This diversion of electron flow will, itself, compromise the biosynthesis of cysteine and methionine from sulfate. Furthermore the product of the dismutation of 02. is 02 t Hz02 and SOD cannot change that. Diminution, or elimination, of SOD will increase sensitivity of cells to oxygen and to compounds which can divert univalent electron flow to oxygen; however, elevation of SOD above wild type levels should therefore not be expected to provide complete protection against overproduction of 02..

1~3 DT-DIAPHORASE ACTIVITY AND QUINONE TOXICITY: PRO- AND ANTIOXIDANT ASPECTS AND THE EFFECT OF SUPER - OXIDE DISMUTASE Enriquc Cadcnas Department of Molecular Pharmacology & Institute for Toxicology, University of Southern California, Los Angeles, CA 90033

Quinones and quinone reductases play a prominent role in determining the pro- and antioxidant state of living cells under both physiological and a variety of pathological conditions. The biological activation of quinones is achieved mainly through one- or two-electron quinone re- ductases. DT-Diaphorase is a unique flavoprotein, whose antioxidant or protective function against quinone toxicity is supposed to be bas- ed on its predominant feature to catalyze two-electron transfers. This notion has been evaluated in terms of the individual chemistry of qui- nones and of the mechanisms by which they are reduced. The antioxi- dant or prooxidant properties of quinones during DT-diaphorase cataly- sis express primarily a logical response to a particular quinone func tional group chemistry. Three types of reactions could be distingui- shed with the following features: DT-diaphorase catalyzes [al the for- mation of redox-stable hydroquinones; [b] the formation of redox-la- bile hydroquinones, the autoxidation of which is prevented by super- oxide dismutase, and [cl the formation of redox-labile hydroquinones, the autoxidation of which is enhanced by superoxide dismutase. In the first instances, the individual activity of DT-diaphorase or its concert- ed activity with superoxide dismutase (involving suppression of Oz.-- dcpcndent chain propagation reactions by dismutase) could be regarded as antioxidant. In the last instance, DT-diaphomse activity bears a pro- oxidant character. This effect is observed mainly with anticancer quin- ones bearing a leaving- or arylating group, such as methyl alkylating naphthoquinones and diaziridinyl benzoquinones, and it is explained in terms of the redox properties of these quinones, which are mainly cen- tered on the semiquinone species. The significance of these findings -especially addressing the relationship between chemotherapeutic modalities and DT-diaphorase activity in cancer cells- remains to be determined.

SUPEROXIDE-SENSITIVE IRON-SULFUR-CONTAINING (DE)HYDRATASES: TARGETS AND MEASURES OF SUPEROXIDE Paul Gardner

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Department of Pediatrics, Pulmonary Division, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA

Superoxide (02-.) reacts with a limited set of biological

targets. Understanding the toxicity of 02-. continues to demand a greater knowledge of these targets. Iron-sulfur- containing (de)hydratases are particularily susceptible to inactivation by 02-.. These sensitive Fe-S enzymes include

the F colj a$-dihydroxyacid dehydratase, B-phosho- gluconate dehydratase, aconitase, fumarases A and B and the mammalian mitochondrial aconitase. 02-. reacts with and inactivates them 6-phosphogluconate dehydratase and

aconitase with reaction rates of 108-log M-‘s-l. Protection by superoxide dismutase (SOD) and by substrates and the dynamic reactivation of the oxidatively-inactivated 4Fe-4S centers with ferrous ions and glutathione limits the fraction of 02..-inactivated enzyme ifjy&~. Measurements of aconitase activity in u and in cultured mammalian cells have demonstrated the feasibility of using the aconitase as a sensitive measure of steady-state levels of 02.. in m or in the mitochondrial matrix under a variety of conditons. The advantages of coordinating M MnSOD synthesis with

[Fe2+] and an oxidative metabolism via the global transcription regulons ti (ferric uptake regulation) and 8& (aerobic respiration control) will be discussed in light of our knowledge of these critical 02..-sensitive Fe-S (de)hydratases.

MECHANISM OF FNDOCENOUS SUPEROXIDE FORMATlON 1N ESCHERfCHIA COLI AND ITS RELATION TO PATTERNS OF SUPEROXIDE DISMUTASE SYNTHESIS. James A. Imlay and Yahya Kargalioglu. Department of Microbiology, University of Ilhnois, Urbana IL, 61801.

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As part of an effort to develop a complete picture of oxidative stress in E. coli, we are working to define the circwnstance and mechanism of endogenous 0~~ production. Experiments in vifro indicated that most 02‘ produced during aerobiosis is made by amoxidation of the respiratory NADH dehydrogenases, while a significant minority evolves from a succinate-reducible dehydrogenase. Surprisingly, all of the succinate-dependent @- is made by fumarate reductase, which is synthesized only in scant amounts during aerobiosis, rather than by its aerobic isozyme, succinate dehydrogenase. Mechanistic studies suggest that the highly efficient formation of 02‘ by reduced fumarate reductase is due to partial electron occupancy of its flavin moiety, conferring semiquinone character and facilitating electron transfer to weak oxidants. This is conducive to its physiological role of fumarate reduction but also permits inadvertent 0~~ formation when oxygen is present. In contrast the redox balance of succinate dehydrogenase shifts the electrons away from the flavin and towards the iron-sulfur clusters. expediting ubiquinone reduction and barring @- formation.

The propensity of fumarate reductase to generate 02. is mitigated by its virtually complete transcriptional repression during aerobiosis. However, when this facuhative anaerobe re-enters an aerobic habitat, an acute flux of @- from extant fumarate reductase ensues. In fact, it is in preparation for this circumstance that the iron SOD isozyme is svnthesized anaerobicallv. Re-oxvaenated cells reouire ore-formed SOD because the 02- otherwise r&idly inactivates’dibydroxyacid dehydratase, which is essential for branched-chain amino-acid biosynthesis, and thereby impedes synthesis of oxygen-inducible defensive enzymes. Thus mutants deficient in anaerobic SOD lag for two hours upon re-oxygenation. ImportantIy, strains lacking anaerobic SOD do not lag if they also lack fumarate reductase, confming the role of the latter enzyme in generating the toxic 02..

Thus patterns of SOD synthesis in E. coli are well-matched with the biological sources and targets of @-. More generally, the tendency of low-potential electron-tmnsport chains to autoxidize demands that facultative bacteria make regulatory and metabolic accomodations to minimize the stress of m-oxygenation. The oxygen intolerance of obligate anaerobes presumably derives from their failure to do so.