Rhizobial iron regulator A (RirA) is a global regulator of iron homeostasis in many nitrogen-fixing Rhizobia and related species of α-proteobacteria. It belongs to the widespread Rrf2 super-family of transcriptional regulators and features three conserved Cys residues that characterise the binding of an iron-sulfur cluster in other Rrf2 family regulators. Here we report biophysical studies demonstrating that RirA contains a [4Fe-4S] cluster， and that this form of the protein binds RirA-regulated DNA， consistent with its function as a repressor of expression of many genes involved in iron uptake. Under low iron conditions， [4Fe-4S] RirA undergoes a cluster conversion reaction resulting in a [2Fe-2S] form， which exhibits much lower affinity for DNA. Under prolonged low iron conditions， the [2Fe-2S] cluster degrades to apo-RirA， which does not bind DNA and can no longer function as a repressor of the cell's iron-uptake machinery. [4Fe-4S] RirA was also found to be sensitive to O， suggesting that both iron and O are important signals for iron metabolism. Consistent with this， data showed that expression of RirA-regulated genes is also affected by O. These data lead us to propose a novel regulatory model for iron homeostasis， in which RirA senses iron the incorporation of a fragile iron-sulfur cluster that is sensitive to iron and O concentrations.