Expression, purification, and characterization of a [Fe2S2] cluster containing ferredoxin from Acidithiobacillus ferrooxidans

Curr Microbiol. 2007 Dec;55(6):518-23. doi: 10.1007/s00284-007-9025-4. Epub 2007 Oct 2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acidithiobacillus thiooxidans / enzymology*
  • Acidithiobacillus thiooxidans / genetics
  • Acidithiobacillus thiooxidans / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Chromatography, Affinity
  • Cloning, Molecular
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Ferredoxins* / chemistry
  • Ferredoxins* / genetics
  • Ferredoxins* / isolation & purification
  • Ferredoxins* / metabolism
  • Iron-Sulfur Proteins* / chemistry
  • Iron-Sulfur Proteins* / genetics
  • Iron-Sulfur Proteins* / isolation & purification
  • Iron-Sulfur Proteins* / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Bacterial Proteins
  • Ferredoxins
  • Iron-Sulfur Proteins