Abstract
Iron regulatory protein 1 (IRP1) regulates the synthesis of proteins involved in iron homeostasis by binding to iron-responsive elements (IREs) of messenger RNA. IRP1 is a cytoplasmic aconitase when it contains a [4Fe-4S] cluster and an RNA-binding protein after complete removal of the metal center by an unknown mechanism. Human IRP1, obtained as the pure recombinant [4Fe-4S] form, is an enzyme as efficient toward cis-aconitate as the homologous mitochondrial aconitase. The aconitase activity of IRP1 is rapidly lost by reaction with hydrogen peroxide as the [4Fe-4S] cluster is quantitatively converted into the [3Fe-4S] form with release of a single ferrous ion per molecule. The IRE binding capacity of IRP1 is not elicited with H(2)O(2). Ferrous sulfate (but not other more tightly coordinated ferrous ions, such as the complex with ethylenediamine tetraacetic acid) counteracts the inhibitory action of hydrogen peroxide on cytoplasmic aconitase, probably by replenishing iron at the active site. These results cast doubt on the ability of reactive oxygen species to directly increase IRP1 binding to IRE and support a signaling role for hydrogen peroxide in the posttranscriptional control of proteins involved in iron homeostasis in vivo.
Highlights
Over the last 10 years, proteins known as iron regulatory proteins (IRPs)1 have been recognized as the major regulatory factors for iron uptake and storage in most mammalian cells [1, 2]
UV-visible spectra of cytoplasmic aconitases purified from natural sources have not been reported, but the visible absorbance of pure bovine mAcn is very similar to that of rhIRP1
Bacteria Can Produce Active Human cAcn—The occurrence and function of iron regulatory proteins have so far only been described for metazoans [2, 31], the presence of mRNA sequences resembling iron-responsive elements (IREs) has been noticed in bacteria [46]
Summary
Over the last 10 years, proteins known as iron regulatory proteins (IRPs) have been recognized as the major regulatory factors for iron uptake and storage in most mammalian cells [1, 2]. IRP1 belongs to an isomerase family of proteins containing one [4Fe-4S] cluster and including mitochondrial aconitase [4]. It exhibits aconitase activity [5]. Low concentrations of H2O2 induce a fast and potent activation of IRP1 in intact cells, but not in lysates (8 –11) The latter results seemingly contradict the notion that reactive oxygen species (ROS) in general, and H2O2 in particular, directly target and destroy iron-sulfur clusters [12, 13]. The efficient heterologous production of human IRP1 (rhIRP1) with its full content of [4Fe-4S] cluster has allowed us to assess the consequences of reactions with oxygen derivatives on the functional properties of the cytoplasmic aconitase
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