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 [Fe(2)S(2)] cluster of the protein.
Zeng
Jia
J
Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha 410083, People's Republic of China. zengjcsu@yahoo.com.cn
Huang
Xia
X
Liu
Yuandong
Y
Liu
Jianshe
J
Qiu
Guanzhou
G
D013856:Acidithiobacillus thiooxidans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins