Expression, Purification, and Characterization of Iron-Sulfur Cluster Assembly Regulator IscR from Acidithiobacillus ferrooxidans

IscR (iron-sulfur cluster regulator) has been reported to be a repressor of the iscRSUA operon, and in vitro transcription reactions have revealed that IscR has a repressive effect oil the iscR promoter in the case of |Fe2S2| cluster loading. In the present study, the iscR gene from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, and then purified by one-step affinity chromatography to homogeneity. The molecular mass of the IscR was 18 kDa by SDS-PAGE. The optical and EPR spectra results for the recombinant IscR confirmed that an iron-sulfur cluster was correctly inserted into the active site of the protein. However, no |Fe2S2| cluster was assembled in apolscR with ferrous iron and sulfide in vitro. Therefore, the |Fe2S2| cluster assembly in IscR in vivo would appear to require scaffold proteins and follow the Isc "AUS" pathway.