The iron-sulfur cluster regulator (IscR) was reported to be a repressor of the iscRSUA operon. In vitro transcription reactions revealed that the IscR had a repression effect on the iscR promoter. The IscR contains a [Fe2S2] cluster per each monomer, and three highly conserved cysteines were identified to ligate the [Fe2S2] cluster. It was proposed that a non-cysteine residue might be the fourth ligand for the [Fe2S2] cluster. In this study, using site-directed mutagenesis, Glu43 was found to be the fourth residue that coordinates the [Fe2S2] cluster of IscR.