Expression, Purification, And The Characterization Of An Iron-Sulfur Cluster Assembly Regulator IscR From Acidithiobacillus Ferrooxidans

The [2Fe-2S] cluster containing IscR 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 IscR from Acidithiobacillus ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The 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 Cys92, Cys99, and Cys107 were ligating with the [2Fe2S] 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. Moreover, 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 IscR 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 [2Fe2S] cluster in IscR from A. ferrooxidans in vitro in the presence of three scaffold proteins of IscA, IscS and IscU. The spectra and EPR results of the holo- IscR confirmed that the iron-sulfur cluster was correctly assembled into the protein. The assembly of [2Fe2S] cluster in IscR in vivo might also follow the isc"AUS" mechanism. Recently report note that the IscR as regulator acted two functionally overlapping Fe-S cluster assembly systems in E. coli in opposite ways under different conditions of oxidative stress.