Acidithiobacillus ferrooxidans ( Af.) is a microorganism of significant biotechnological interest that thrives in acidic environments with very high concentrations of soluble iron. Understanding the molecular mechanisms that enable its survival in these extreme conditions is of great scientific relevance and practical importance for bioleaching of precious metals. Central to its metabolism is the Ferric Uptake Regulator (Fur), a protein that regulates iron homeostasis and responses to oxidative stress in bacteria. Using a combination of bioinformatics, experimental, and spectroscopic methodologies, this study identified and characterized the metal binding sites and cofactors relevant to AfFur´s function. Three metal-binding sites became evident, two of which are very similar to those found in other members of the superfamily. The third site, formed by four cysteine residues in an configuration (CX 2C-X n-CX 8C), stably binds a iron-sulfur cluster. Site-directed mutagenesis coupled with Electrophoretic Mobility Shift Assays underscored the essentiality of these cysteines for AfFur’s functionality, particularly in DNA binding. Altogether, the findings from this research suggest the presence of an iron-sulfur cluster, which may play a role in fine-tuning iron sensing, particularly adapted to the unique environment of Acidithiobacillus ferrooxidans.