Peroxynitrite and nitric oxide differently target the iron-sulfur cluster and amino acid residues of human iron regulatory protein 1

被引:44
作者
Soum, E
Brazzolotto, X
Goussias, C
Bouton, C
Moulis, JM
Mattioli, TA
Drapier, JC [1 ]
机构
[1] CNRS, Inst Chim Subst Nat, F-91190 Gif Sur Yvette, France
[2] CEA, Dept Reponse & Dynam Cellulaires, F-38054 Grenoble, France
[3] CEA Saclay, Dept Biol Joliot Curie, Sect Bioenerget, Lab Biophys Stress Oxydant, F-91191 Gif Sur Yvette, France
关键词
D O I
10.1021/bi030041i
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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]11 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.
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页码:7648 / 7654
页数:7
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