Expression, Purification, And Characterization Of A [Fe₂S₂] Cluster Containing Ferredoxin From Acidithiobacillus Ferrooxidans

The [2Fe-2S] cluster containing ferredoxin has attracted much attention in recent years. Genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins and functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. The gene of ferredoxin from Acidithiobacillus ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The MALDI-TOF MS and spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys42, Cys48, Cys51, and Cys87 were ligating with the [Fe₂S₂] cluster of the protein.Iron-sulfur [Fe-S] clusters are one of the most ubiquitous and functionally versatile prosthetic groups in nature. Clusters contain [2Fe-2S], [3Fe-4S], [4Fe-4S], or [8Fe-7S] core units. Different types of biological [Fe-S] clusters have different functions, including electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. [Fe-S] clusters could be incorporated into their protein partners both in vivo and in vitro. Three different types of [Fe-S] cluster biosynthetic systems have been discovered. Although most of the key players participating in biosynthesis of [Fe-S] cluster have been identified, the molecular mechanism is still unknown. Moveover, how the different proteins interact with each other to accomplish [Fe-S] protein maturation in vivo and how to faithfully duplicate that process in vitro still need to further study.The [2Fe-2S] cluster containing ferredoxin plays an important role in the maturation of various iron-sulfur (Fe-S) proteins, and functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. We report here the assembly of [Fe₂S₂] cluster in ferredoxin from A. ferrooxidans in vitro in the presence of three scaffold proteins of IscA, IscS and IscU. The UV-vis spectra and EPR results of the holo-ferredoxin confirmed that the iron-sulfur cluster was correctly assembled into the protein. The assembly of [Fe₂S₂] cluster in ferredoxin in vivo might also follow the ISC'AUS" mechanism.