Mechanisms of iron- and O 2 -sensing by the [4Fe-4S] cluster of the global iron regulator RirA.

RirA is a global regulator of iron homeostasis in Rhizobium and related aproteobacteria. 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 O-2. The data indicate that the key iron-sensing step is the O-2 -independent, reversible dissociation of Fe2+ from [4Fe-4S](2+) to form [3Fe-4S](0). The dissociation constant for this process was determined as K-d = similar to 3 mu M, which is consistent with the sensing of 'free' iron in the cytoplasm. O-2-sensing occurs through enhanced cluster degradation under aerobic conditions, via O-2-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.