The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was predicted to have novel features that work in reverse to catalyse the sulfane sulfur of GSnH species (n > 1) into sulfite in sulfur oxidation. There are two different highly upregulated genes potentially encoding the HdrC subunit in A. ferrooxidans grown in sulfur-containing medium. An HdrC containing iron-sulfur cluster from A. ferrooxidans corresponding to one of the genes had been expressed and biophysically characterized. Comparatively, here we report the cloning, expression, and characterization of another HdrC from A. ferrooxidans. This HdrC was expressed in inclusion bodies in all conditions tested. This purified HdrC displayed brown color and contained the [4Fe-4S] cluster confirmed by the UV-scanning and EPR spectra. This HdrC owned two identical motifs (Cx(2)Cx(2)Cx(3)C) including total of eight cysteine residues for [4Fe-4S] cluster binding. To our surprise, the site-directed mutagenesis results of these eight residues revealed that respective removal of the sulfhydryl group of Cys73, Cys76, Cys79, and Cys37 resulted in the cluster loss, but those of Cys27, Cys30, Cys33, and Cys83 had no influence, which demonstrated that this HdrC bound only one cluster, and it might be responsible for causing the HdrABC in A. ferrooxidans working in reverse. Molecular modeling results also supported the above results and showed that this cluster was ligated by Cys73, Cys76, and Cys79 in one motif and Cys37, however, in another motif.