| Abstract
| - Iron regulatory protein 1 (IRP1) is a redox-sensitive protein which exists in two active formsin the cytosol of eukaryotic cells. Holo-IRP1 containing a [4Fe-4S] cluster exhibits aconitase activitywhich catalyzes the isomerization of citrate and isocitrate. The cluster-free protein (apo-IRP1) is atransregulator binding to specific mRNA, and thus post-transcriptionally modulating the expression ofgenes 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 stretchingmode 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 electronparamagnetic resonance (EPR) data and correlated with the loss of aconitase activity. In another series ofexperiments, the RR spectra also revealed the presence of additional bands at 818 and 399 cm-1 whenrhIRP1 was treated with a peroxynitrite synthesized by a different procedure. These bands correspond tothose of 3-nitrotyrosine, and they indicate nitration of at least one tyrosine residue in rhIRP1. This wasfurther confirmed by Western blot analysis with an anti-nitrotyrosine antibody. In contrast, the reactionof rhIRP1 with NO in the absence of oxygen revealed full mRNA binding activity of the protein, withoutnitration of tyrosines. These results strongly suggest that NO mainly acts as a regulator of IRP1 whereasperoxynitrite is likely to disrupt the IRP1/IRE regulatory pathway.
|
