Mechanisms of iron- and O₂-sensing by the [4Fe-4S] cluster of the global iron regulator RirA.

RirA is a global regulator of iron homeostasis in <i>Rhizobium</i> and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses iron uptake by binding to IRO Box sequences upstream of RirA-regulated genes. Under low iron and/or aerobic conditions, [4Fe-4S] RirA undergoes cluster conversion/degradation to apo-RirA, which can no longer bind IRO Box sequences. Here, we apply time-resolved mass spectrometry and electron paramagnetic resonance spectroscopy to determine how the RirA cluster senses iron and O₂. The data indicate that the key iron-sensing step is the O₂-independent, reversible dissociation of Fe²⁺ from [4Fe-4S]²⁺ to form [3Fe-4S]⁰. The dissociation constant for this process was determined as <i>K</i>_d = ~3 µM, which is consistent with the sensing of 'free' iron in the cytoplasm. O₂-sensing occurs through enhanced cluster degradation under aerobic conditions, via O₂-mediated oxidation of the [3Fe-4S]⁰ intermediate to form [3Fe-4S]¹⁺. This work provides a detailed mechanistic/functional view of an iron-responsive regulator.