Human cytoplasmic aconitase (iron regulatory protein 1) is converted into its [3Fe-4S] form by hydrogen peroxide in vitro but is not activated for iron-responsive element binding

Iron regulatory protein 1 (IRP1) regulates the synthesis of proteins involv ed in iron homeostasis by binding to iron-responsive elements (IREs) of mes senger RNA. IRP1 is a cytoplasmic aconitase when it contains a [4Fe-4S] clu ster 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 c onverted into the [3Fe-4S] form with release of a single ferrous ion per mo lecule. The IRE binding capacity of IRP1 is not elicited with H2O2. Ferrous sulfate (but not other more tightly coordinated ferrous ions, such as the complex with ethylenediamine tetraacetic acid) counteracts the inhibitory a ction of hydrogen peroxide on cytoplasmic aconitase, probably by replenishi ng iron at the active site. These results cast doubt on the ability of reac tive 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.