IscA was proposed to be involved in the iron-sulfur cluster assembly encoded by the iscSUA operon, but the role of IscA in the iron-sulfur cluster assembly still remains controversial. In our previous study, the IscA from A. ferrooxidans was successfully expressed in Escherichia coli, and purified to be a [Fe4S4]-cluster-containing protein. Cys35, Cys99, and Cys101 were important residues in ligating with the [Fe4S4] cluster. In this study, Asp97 was found to be another ligand for the iron-sulfur cluster binding according to site-directed mutagenesis results. Molecular modeling for the IscA also showed that Asp97 was a strong ligand with the [Fe4S4] cluster, which was in good agreement with the experimental results. Thus, the [Fe4S4] cluster in IscA from A. ferrooxidans was ligated by three cysteine residues and one aspartic acid.