Iron regulatory protein (IRP) is a cytosolic bifunctional [Fe-S] protein which exhibits aconitase activity or binds iron responsive elements (IREs) in untranslated regions of specific mRNA. The modulators of these activities are the intracellular concentration of iron and, as recently described, NO synthase activity. In this study, we attempted to establish in in vitro experiments whether peroxynitrite (ONOO
Iron regulatory protein (IRP)1 controls iron homeostasis by regulating the expression of ferritin and transferrin receptor through binding to iron-responsive elements (IREs) in the 5Ј-and 3Ј-untranslated regions of their respective mRNAs. In iron-depleted cells, IRP has high affinity for IRE(s) and both stabilizes transferrin receptor mRNA and prevents ferritin translation. Conversely, in iron-replenished cells, it functions as an [Fe-S] cluster-containing aconitase which converts citrate into isocitrate in the cytosol (1, 2). These activities are mutually exclusive, because the fully assembled [4Fe-4S] cluster required for enzymatic activity at the active site sterically inhibits IRE binding (2, 3). We previously reported that induction of nitric-oxide synthase in activated murine macrophages results in loss of mitochondrial enzyme activity (4, 5). In addition, we showed that inhibition of mitochondrial aconitase was rapidly reversible (6). Subsequently, we and others demonstrated that NO synthesis increases IRE binding in several cell types (7-9). Conversely, NO synthesis causes loss of aconitase activity of IRP as shown for mitochondrial aconitase (7, 9). Therefore, the functional behavior of IRP, either aconitase or IRE-binding protein, can be determined by the intracellular iron concentration (1, 2) and the NO flux into the cell (7-10). Another line of research showed previously that the activities of bacterial and mammalian aconitases are inhibited by O 2 . (11-13), whereas the activity of plant aconitase which exhibits a strong homology with mammalian IRP (14) and ONOO Ϫ . For this purpose, we compared the direct effect of these three species on IRP activities to that of various NO donors on IRP activities. The results showed that even though ROS and ONOO Ϫ inhibited aconitase activity of IRP, contrary to NO donors, they did not activate its IRE binding activity.
EXPERIMENTAL PROCEDURESMaterials-S-Nitroso-L-glutathione (GSNO) was purchased from Alexis (Switzerland). S-Nitrosothiol derived from cysteamine and referred to as S-nitrosocysteamine (SNC) was kindly provided by Pr. Marc Fontecave, Grenoble, France. Spermine-NONOate (Sper/NO) was from Cayman Chemical Co. (Ann Arbor, MI). Dihydrorhodamine was purchased from Molecular Probes, Leiden, Holland. 3-Morpholinosydnonimine hydrochloride (SIN-1) and its end product SIN-1C were synthesized by Cassella AG (Frankfurt, Germany) and kindly provided by J. Winicki, Laboratoires Hoechst, France. Hydrogen peroxide was from Merck (Darmstadt, Germany). Xanthine oxidase (720 milliunits/mg), catalase, bovine erythrocyte Cu,Zn-superoxide dismutase (SOD), bovine he...