Superoxide Radical and Iron Modulate Aconitase Activity in Mammalian Cells

Abstract

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress

“…Gardner & Fridovich (1992) and Gardner et al (1995) reported first that mammalian aconitase was inhibited by superoxide and later it has been shown that nitric oxide (Andersson et al 1998), peroxynitrate (Andersson et al 1998) and, as shown by our group, H 2 O 2 (Chinopoulos et al 1999; also inhibited this enzyme, most likely due to interaction with the iron-sulphur clusters. Generally, iron-sulphur centres of proteins are primary sites for ROS to attack mitochondrial enzymes and this may be the mechanism by which succinate dehydrogenase is also inhibited by ROS.…”

Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

“…Although tyrosine nitration of PDHC may account for postischemic alterations in metabolism, parallel changes resulting from the effects of peroxynitrite on other cellular components could also contribute to inhibition of cerebral energy metabolism. For example, oxidative stress can damage other proteins, including TCA cycle enzymes [61,64]. Moreover, electron transport chain Complexes I and II are also very sensitive to inhibition caused by reactive oxygen and nitrogen species, including peroxynitrite [65].…”

The influence of nickel and cobalt on putative members of the oxygen‐sensing pathway of erythropoietin‐producing HepG2 cells

“…One-electron oxidation of the SoxR (2Fe-2 S) clusters is the mechanism both for sensing oxidative stress and for activating the SoxR protein as a transcription factor [41]. The inactivation-reactivation of aconitase in cultured mammalian cells depends on oxygen radicals with an aconitase 50% active at a O Ϫ 2 concentration of 50Ϫ200 pM and 86% active at a O Ϫ 2 concentration of 8Ϫ30 pM [42]. The active RNA-binding form of the aconitase without 4Fe-4 S cluster binds to the 3′ iron responsive element of the transferrin receptor (TfR) mRNA and increases its stability.…”