RESUMO
Iron regulatory proteins (IRPs) control iron metabolism in mammalian cells by binding to the iron-responsive element (IRE) in the target mRNA. Heme regulatory motifs (HRMs) are conserved in the two IRP homologues IRP1 and IRP2 that specifically bind to two and three heme equivalents, respectively; however, only the heme binding to the iron-dependent degradation (IDD) domain of IRP2 causes heme-mediated oxidation, which does not occur in IRP1. Therefore, the functional significance of conserved HRMs outside the IDD domain is yet unclear. In this study, spectroscopic heme titration with IRP mutants confirmed heme binding to each HRM in IRPs, and the effect of heme binding to HRMs on IRE binding was examined. Native polyacrylamide gel electrophoresis analysis revealed that heme binding to HRMs near the IRE binding site inhibits complex formation between IRPs and IRE without oxidative modification, indicating that the function of HRMs varies outside and within the IDD domain. However, the formation of a typical reactive oxygen species (ROS), hydrogen peroxide, was spectroscopically detected in both heme-bound IRPs. Comparing the heme environmental structures surrounding HRMs, the flexible conformation and many amino acid residues sensitive to ROS of the IDD domain were suggested to promote specific oxidation by the generated hydrogen peroxide. Thus, heme binding to HRM near the IRE binding site sterically interferes with IRE binding, while HRM in the IDD domain facilitates specific heme-mediated oxidation of the protein moiety and the protein degradation via the ubiquitin-proteasome system, resulting in the inhibition of IRE binding.