Nitric oxide and peroxynitrite promote complete disruption of the [4Fe-4S] cluster of recombinant human iron regulatory protein 1.
Iron regulatory protein 1 (IRP1) is a metalloprotein which regulates several proteins involved in mammalian iron homeostasis at a post-transcriptional level, by binding to specific mRNA sequences termed iron responsive elements (IREs). IRP1 exhibits two mutually exclusive activities, either aconitase or mRNA-binding protein, depending on the intactness of a versatile [4Fe-4S] cluster. Here we asked whether NO and peroxynitrite act directly on IRP1 and how they modify its functions. Recombinant human IRP1 was purified from Escherichia coli as a [4Fe-4S] cluster-containing protein, and exposed to 3-morpholinosydnonimine (SIN-1), which was used either as a peroxynitrite donor or as an NO donor when added with an excess of superoxide dismutase (SOD). We showed that, in both settings, IRP1 lost aconitase activity and iron concomitantly. Iron release reached 3.5-4 iron atoms per IRP1 molecule, proving that the Fe-S cluster was completely disrupted. An increase in IRP1 IRE-binding was observed upon the sequential addition of SIN-1/SOD and low concentrations of 2-mercaptoethanol, whereas SIN-1 alone induced a decrease in binding capacity which was not reversed by 2-mercaptoethanol, even at high concentrations. Moreover, nitrotyrosine adducts were detected on SIN-1-treated IRP1 by Western blot analysis.
Soum
Emmanuelle
E
Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, avenue de la Terrasse, 91190 Gif-sur-Yvette, France.
Drapier
Jean-Claude
JC
D000154:Aconitate Hydratase / Q000378:metabolism; D015153:Blotting, Western; D017124:Conserved Sequence; D024202:Electrophoretic Mobility Shift Assay; D005297:Ferrozine / Q000737:chemistry; D006801:Humans; D035941:Iron Regulatory Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D009566:Nitrates; D011994:Recombinant Proteins; D030421:Peroxynitrous Acid; D005297:Ferrozine; D009569:Nitric Oxide; D014443:Tyrosine; C002385:linsidomine; D008623:Mercaptoethanol; D008981:Molsidomine; D013482:Superoxide Dismutase; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1