Cloning, Overexpression and Characterization of the FeSI Protein from Azotobacter vinelandii CA6

Abstract

The [2Fe-2S] protein from Azotobacter vinelandii CA6, also known as Shethna protein I or FeSI protein, was cloned and overexpressed in E. coli and purified. SDS-PAGE analysis showed a band at ~11 kDa, the monomeric size of the protein, at each stage of the purification. Gel filtration profile of FeSI indicates it forms a dimer in its native state. The UV-visible spectrum showed absorbances at signature wavelengths, 344, 418 and 464 nm, due to the iron-sulfur cluster. The sequence of A. vinelandii CA6 FeSI protein are similar to the sequences of [2Fe-2S] ferredoxins from nitrogen-fixing Clostridium pasteurianum and Aquifex aeolicus, which is not a nitrogen fixer, including conserved cysteine residues. These suggest that FeSI may or may not be involved in nitrogen fixation as there is no evidence although the FeSI gene is present in the major nif gene cluster in Azotobacter vinelandii CA6. This study will be beneficial for understanding the function of FeSI in nitrogen fixation and the relations with other [2Fe-2S] proteins.