| Heme is an essential molecule involved in many cellular functions. The heme biosynthetic pathway is coordinated with iron availability and other variables to accommodate the cellular demand for heme. The iron response regulator (Irr) protein mediates iron control of the heme pathway in Bradyrhizobium japonicum. Irr is a conditionally stable protein that accumulates under iron limitation but degrades in response to iron. This turnover is heme-dependent and rapid degradation involves heme binding to a heme regulatory motif (HRM). Here, by analyzing Irr truncation and mutation derivatives, we identified a second heme binding site that is necessary for degradation. The HRM and the new heme site bound the ferric and ferrous forms of heme, respectively, showing that normal Irr degradation involves both redox states of heme. Irr degradation required oxygen in vivo. Furthermore, heme-dependent Irr oxidation was demonstrated in vitro. A truncated Irr protein unable to bind ferrous heme was not oxidized in vitro and did not degrade in vivo. The results suggested that protein oxidation is a signal for Irr degradation. Moreover, hydrogen peroxide accelerated Irr turnover and elevated heme biosynthetic enzyme ALAD levels in vivo, as well as promoted Irr oxidation in vitro. The findings suggested that cells sense and respond to oxidative stress through Irr to regulate heme biosynthesis.;Whole genome microarray analysis showed that Irr controls many iron-regulated genes, and not only those involved in heme biosynthesis. We showed that Irr directly binds to ICE-like motifs upstream of regulated genes and can have both a positive and negative activity. These findings suggested that B. japonicum senses iron availability via the status of heme biosynthesis to regulate iron homeostasis and metabolism. Additionally, microarray analysis showed that B. japonicum Fur is involved in iron regulation; however, it has novel roles compared to Fur from other bacteria. Collectively, we propose that Irr, rather than Fur, is a global iron regulator in B. japonicum. |
