Expression, purification and molecular modelling of the Iro protein from Acidithiobacillus ferrooxidans Fe-1.
The Iro protein was proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, it is a member of HiPIP family with the iron-sulfur cluster for electron transfer. The gene of Iro protein from A. ferrooxidans Fe-1 was cloned and then successfully expressed in Escherichia coli, finally purified by one-step affinity chromatography to homogeneity. The recombinant protein was observed to be dimer. The molecular mass of a monomer containing the [Fe4S4] cluster was 6847.35 Da by MALDI-TOF-MS. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Molecular modelling for the protein revealed that Cys20, Cys23, Cys32 and Cys45 were in ligation with the iron-sulfur cluster, and Tyr10 was important for the stability of the [Fe4S4] cluster. As we know, this is the first report of expression in E. coli of the Iro protein from A. ferrooxidans Fe-1.
Zeng
Jia
J
Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha 410083, PR China.
Geng
Meimei
M
Liu
Yuandong
Y
Zhao
Wenjie
W
Xia
Lexian
L
Liu
Jianshe
J
Qiu
Guanzhou
G
D013856:Acidithiobacillus thiooxidans / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D010088:Oxidoreductases; C074859:iron oxidase, Thiobacillus ferrooxidans