Nitrogen monoxide activates iron regulatory protein 1 RNA-binding activityby two possible mechanisms: Effect on the [4Fe-4S] cluster and iron mobilization from cells

The iron-regulatory protein 1 (IRP1) regulates the expression of several mo lecules involved in iron (Fe) metabolism by reversibly binding to iron-resp onsive elements (IREs) in the untranslated regions (UTR) of particular mRNA transcripts. Several studies have indicated chat nitrogen monoxide (NO) ma y influence IRP1 RNA-binding activity by a direct effect on the [4Fe-4S] cl uster of the protein. It has also been suggested that NO may act indirectly on IRP1 by affecting the intracellular Fe pools that regulate the function of this protein [Pantopoulous et al. (1996) Mol. Cell. Biol. 16, 3781-3788 ]. There is also the possibility that NO may S-nitrosate sulfhydryl groups that are crucial for mRNA binding and decrease IRP1 activity by this mechan ism. We have examined the effect of a variety of NO donors [e.g., S-nitroso -N-acetylpenicillamine (SNAP), spermine-NONOate (SperNO), and S-nitrosoglut athione (GSNO)] on IRP1 RNA-binding activity in bath LMTK- fibroblast lysat es and whole cells. In cell lysates, the effects of NO at increasing RNA-bi nding activity were only observed when cells were made Fe-replete. Under th ese circumstances, IRP1 contains an [4Fe-4S] cluster that was susceptible t o NO. In contrast, when lysates were prepared from cells treated with the F e chelator desferrioxamine (DFO), NO had no effect on the RNA-binding activ ity of IRP1. The lack of effect of NO under these conditions was probably b ecause this protein does not have an [4Fe-4S] cluster. In contrast to the N O generators above, sodium nitroprusside (SNP) decreased IRP1 RNA binding w hen cells were incubated with this compound. However, SNP had no effect on IRP1 RNA-binding activity in lysates, suggesting that the decrease after in cubation of cells with SNP was not due to S-nitrosation of critical sulfhyd ryl groups. Apart from the direct effect of NO on IRP1 in Fe-replete cells, we have shown that NO generated by SNAP, SperNO, and GSNO could also mobil ize Fe from cells. When NO generation was induced in RAW 264.7 macrophages, an increase in IRP1 RNA-binding activity occurred but there was only a sma ll increase in Fe release. Our results suggest that NO could activate IRP1 RNA-binding by two possible mechanisms: (I) its direct effect on the [4Fe-4 S] cluster and (2) mobilization of Fe-59 from cells resulting in Fe depleti on, which then increases IRP1 RNA-binding activity.