Modulation of iron regulatory protein functions - Further insights into the role of nitrogen- and oxygen-derived reactive species

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-, the product of the reaction between NO and O-2(radical anion)), as well as oxygen-derived radicals (O-2(radical anion) and H2O2) and various NO donors, allow IRP to bind IREs using cytosol extract of macrophage-like RAW 264.7 cells. Neither the addition of a bolus of ONOO- or H2O2 nor O-2(radical anion) generation significantly affected IRE binding even though they inhibited its aconitase activity. Moreover, we show that 3-morpholinosydnonimine (SIN-1), a chemical which releases both NO and O-2(radical anion), enhanced IRE binding activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus glutathione (GSH) activated IRE binding by IRP whereas oxyhemoglobin prevented enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, an aconitase substrate, also abolished the effect of SIN-1/SOD on IRE binding by IRP. These results imply that neither O-2(radical anion) nor ONOO- can convert [4Fe-4S] IRP into IRE-binding protein but rather suggest that an active redox form of NO converts IRP into its IRE binding form by targeting the [Fe-S] cluster.