Identification of a Heme-sensing Domain in Iron Regulatory Protein 2

Abstract

Iron regulatory protein 2 coordinates the cellular regulation of iron metabolism by binding to iron-responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. The mechanisms that prevent degradation during iron deficiency or promote degradation during iron sufficiency are not delineated. Iron regulatory protein 2 contains a domain not present in the closely related iron regulatory protein 1, and we found that this domain binds heme with high affinity. A cysteine within the domain is axially liganded to the heme, as occurs in cytochrome P450. The protein-bound heme reacts with molecular oxygen to mediate the oxidation of cysteine, including ␤-elimination of the sulfur to yield alanine. This covalent modification may thus mark the protein molecule for degradation by the proteasome system, providing another mechanism by which heme can regulate the level of iron regulatory protein 2.

Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein

“…Clear evidence was obtained indicating a specific, but probably not quantitative, interaction between haem and a cysteine residue of the truncated peptides, possibly Cys 201 of the Cys-Pro-Phe-His motif. In contrast, full-length 73aa-Domain does not provide specific ligands to haem, in apparent contradiction to previous conclusions [16][17][18][19]. Yamanaka et al [17] showed that the absorption spectrum of haem incubated with purified His-tagged IRP2 had a maximum at 420 nm; this result was used as evidence for haem binding to IRP2, but was later dismissed as being contributed by the His tag [19].…”

“…This would indicate that the peptide still binds haem in the absence of all cysteine residues; otherwise the spectrum should be blank in the visible region ( Figure 5, dotted spectra). Instead of showing that one of the three cysteine residues of the peptide is a haem-binding site [18], these results are rather indicative of weak interactions between haem and the 63-amino-acid peptide, the specificity of which remains to be established.…”

“…Yamanaka et al [17] showed that the absorption spectrum of haem incubated with purified His-tagged IRP2 had a maximum at 420 nm; this result was used as evidence for haem binding to IRP2, but was later dismissed as being contributed by the His tag [19]. Jeong et al [18] reported that the absorption spectrum of a peptide from the specific IRP2 domain showed two maxima upon incubation with haemin, at 370 and 420 nm. The material used in the present study was a ∼ 63-amino-acids peptide spanning the first residue encoded by exon 5 of the human IRP2 gene up to Leu 200 .…”

“…It has been established that iron accelerates IRP2 degradation [14,31] and it has been proposed that IRP2 detects intracellular iron levels by binding haem via the 73aa-Domain [17][18][19] in a process triggering its degradation [19,32]. Since the cleavage sites shown in Figure 1(A) leave truncated fragments containing all four cysteine residues and the histidine of the 73aa-Domain, but none of the lysine residues, it was decided to investigate haem and metal binding to these peptides.…”

“…Early work led to the conclusion that a prolineand cysteine-rich stretch of 73 amino acids [73aa-Domain (73-amino-acid domain)] encoded by the unique IRP2-specific exon 5 is necessary and sufficient for IRP2 degradation by a mechanism involving oxidation of cysteine residues upon iron binding and polyubiquitination [14,16]. More recent results have proposed that the 73aa-Domain plays a role in IRP2 degradation in response to haem and that the cysteine residue of a Cys-Pro-Phe-His, haem regulatory-like, motif in the domain binds oxidized haem [17][18][19]. However, studies by independent groups showed that an IRP2 deletion mutant lacking the full-length domain remains sensitive to iron-dependent degradation, similar to wild-type IRP2 [20,21].…”