Electron and Proton Transfers Modulate DNA Binding by the Transcription Regulator RsrR.

The [Fe2S2]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed (Out) and buried (In) states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the Out and In states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the In state due to a change in its pKa caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes. Our findings illustrate a novel function of protonation resulting from electron transfer.

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 Rhizobium 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 Kd = ~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.

Sensing iron availability via the fragile [4Fe-4S] cluster of the bacterial transcriptional repressor RirA.

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 O2, suggesting that both iron and O2 are important signals for iron metabolism. Consistent with this, in vivo data showed that expression of RirA-regulated genes is also affected by O2. These data lead us to propose a novel regulatory model for iron homeostasis, in which RirA senses iron via the incorporation of a fragile iron-sulfur cluster that is sensitive to iron and O2 concentrations.