Peroxynitrite and nitric oxide differently target the iron-sulfur cluster and amino acid residues of human iron regulatory protein 1.
Iron regulatory protein 1 (IRP1) is a redox-sensitive protein which exists in two active forms in the cytosol of eukaryotic cells. Holo-IRP1 containing a [4Fe-4S] cluster exhibits aconitase activity which catalyzes the isomerization of citrate and isocitrate. The cluster-free protein (apo-IRP1) is a transregulator binding to specific mRNA, and thus post-transcriptionally modulating the expression of genes involved in iron metabolism. The resonance Raman (RR) spectra of human recombinant holo-IRP1 (rhIRP1) excited at 457.9 nm show that the 395 cm(-1) band, attributed to a terminal Fe-S stretching mode of the cluster, is replaced by a 405 cm(-1) band, consistent with the conversion of the [4Fe-4S](2+) center to a [3Fe-4S](+) center, upon exposure to peroxynitrite. This conclusion was confirmed by electron paramagnetic resonance (EPR) data and correlated with the loss of aconitase activity. In another series of experiments, the RR spectra also revealed the presence of additional bands at 818 and 399 cm(-1) when rhIRP1 was treated with a peroxynitrite synthesized by a different procedure. These bands correspond to those of 3-nitrotyrosine, and they indicate nitration of at least one tyrosine residue in rhIRP1. This was further confirmed by Western blot analysis with an anti-nitrotyrosine antibody. In contrast, the reaction of rhIRP1 with NO in the absence of oxygen revealed full mRNA binding activity of the protein, without nitration of tyrosines. These results strongly suggest that NO mainly acts as a regulator of IRP1 whereas peroxynitrite is likely to disrupt the IRP1/IRE regulatory pathway.
Soum
Emmanuelle
E
Institut de Chimie des Substances Naturelles, CNRS, 91190 Gif-sur-Yvette, France.
Brazzolotto
Xavier
X
Goussias
Charilaos
C
Bouton
Cécile
C
Moulis
Jean-Marc
JM
Mattioli
Tony A
TA
Drapier
Jean-Claude
JC
D000154:Aconitate Hydratase / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D035941:Iron Regulatory Protein 1 / Q000737:chemistry / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013059:Spectrum Analysis, Raman; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D013455:Sulfur; D007501:Iron; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1