Cluster occupancy- and oxidation state-dependence of Yersinia enterocolitica IscR DNA binding

Iron‑sulfur (Fe-S) clusters are protein cofactors essential for life. Their assembly requires dedicated cellular machineries, such as the ISC system, found in Escherichia coli and many other bacteria. ISC is regulated by IscR, a member of the Rrf2 family of transcriptional regulators. E. coli IscR (EcIscR) binds a [2Fe-2S] cluster and, in this form, functions as a repressor of the isc operon. Under aerobic conditions there is an increased cellular demand for Fe-S clusters, and apo IscR accumulates resulting in upregulation of ISC. Currently, the signal that EcIscR directly responds to is not clear. Little is known about other IscR homologs and whether key functional features of the E. coli protein are broadly shared. Here, we report studies of the IscR homolog from the pathogen Yersinia enterocolitica. Y. enterocolitica IscR (YeIscR) is ∼80 % identical to EcIscR and binds a [2Fe-2S] cluster most likely coordinated by three conserved Cys residues and one His. Isolated in the 1+ oxidation state, exposure to O₂ or other oxidants resulted in rapid oxidation of the cluster to the +2 state and slow cluster loss. The cluster was relatively insensitive to iron chelators, indicating that it is not labile. While the trigger for degradation of the YeIscR cluster to generate the apo form is not clear, loss of the cluster resulted in a ∼10-fold decrease in DNA affinity. The oxidation state of the cluster was found to be important for DNA binding, with a significant reduction in IscR-bound DNA observed upon oxidation, suggesting possible physiological importance.