SUPEROXIDE RADICAL AND IRON MODULATE ACONITASE ACTIVITY IN MAMMALIAN-CELLS

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O-2(radical anion))-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 O-2(radical anion) 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 t(1/2), of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O-2(radical anion). The ability of ubiquinol-cytochrome c oxidoreductase-generated O-2(radical anion) to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O-2(radical anion) 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 O-2(radical anion)-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 O-2(radical anion).