Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator

The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O(2) to form highly reactive oxygen species (ROS) with the “active site conversion” from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H(2)O(2) from O(2) as found for other hemoproteins, but H(2)O(2) is less effective in oxidizing the peptide, and further activation of H(2)O(2) is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel “two-step self-oxidative modification” mechanism, during which O(2) is activated to form H(2)O(2) at the heme regulatory motif (HRM) site and the generated H(2)O(2) is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.