Jianshe Liu

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 A. 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.

Expression, purification and characterization of an iron-sulfur cluster assembly protein, IscU, from Acidithiobacillus ferrooxidans.

An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe2S2] cluster could be assembled in apoIscU with Fe2+ and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize l-cysteine and Fe2+ to synthesize [Fe2S2] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe2S2] cluster.

In vitro assembly of [Fe4S4] cluster in high potential iron-sulfur protein from Acidithiobacillus ferrooxidans.

High-potential iron–sulfur protein (HiPIP) has been proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, which contains an [Fe4S4] cluster. We report here the assembly of an [Fe4S4] cluster in HiPIP from A. ferrooxidans ATCC 23270 in vitro in the presence of Fe2+ and sulfide. The spectra and matrix-assisted laser desorption ionization–time of flight mass spectrometry results of holoHiPIP confirmed that the iron–sulfur cluster was correctly assembled into the protein.