[Objective] Iron sulfur cluster are one of the most ancient and ubiquitous redox centre in almost all living organism and play an important role in photosynthesis, respiration and nitrogen fixation. [Methods] In this study, three key proteins (IscS, IscU, IscA) involved in iron sulfur cluster assembly from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli. [Results] IscS is a cysteine desulfurase, IscU is a scaffold protein, IscA has strong iron binding activity which acts an iron chaperon. [Conclusion] The Fe-IscA can provide iron for the assembly of transient iron sulfur cluster in IscU in the presence of IscS and L-cysteine in vitro.