A [2Fe-2S] protein encoded by an open reading frame upstream of the Escherichia coli bacterioferritin gene

Biochemistry. 1996 May 21;35(20):6297-301. doi: 10.1021/bi9600862.


An open reading frame located upstream of the bacterioferritin gene in Escherichia coli encodes a hypothetical 64-residue protein [Andrews, S.C., Harrison, P.C., & Guest, J.R. (1989) J. Bacteriol. 171, 3940-3947)]. The spacing of the four cysteine residues in this hypothetical protein is identical to that in a region of NIFU, a [2Fe-2S] protein found in nitrogen-fixing bacteria [Fu, W., Jack, R.F., Morgan, T.V., Dean, D.R., & Johnson, M.K. (1994) Biochemistry 33, 13455-13463)]. The NIFU-like E. coli gene was cloned and overexpressed with a C-terminal "His tag" in E. coli using the T7 RNA polymerase/promoter system, and the protein was purified by metal-chelate affinity chromatography. UV-vis absorption and EPR spectra together with iron and amino acid analyses conclusively established that this NIFU-like E. coli protein contains one [2Fe-2S] cluster which can exist in at least two oxidation levels: +2 for the as-purified protein, and +1 for dithionite-reduced protein. Size-exclusion chromatography established that this His-tagged [2Fe-2S] protein is monomeric in solution. Affinity chromatography demonstrated specific complex formation between bacterioferritin (Bfr) and this NIFU-like [2Fe-2S] protein, which is dubbed Bfd. An open reading frame encoding a homologous Bfd is located near a Bfr gene in at least one other bacterium. Bfd may, therefore, constitute a general redox and/or regulatory component participating in the iron storage or mobilization functions of Bfr.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Base Sequence
  • Cytochrome b Group / chemistry
  • Cytochrome b Group / genetics*
  • DNA Primers / genetics
  • Escherichia coli / chemistry
  • Escherichia coli / genetics*
  • Ferritins / chemistry
  • Ferritins / genetics*
  • Iron-Sulfur Proteins / chemistry
  • Iron-Sulfur Proteins / genetics*
  • Molecular Sequence Data
  • Molecular Structure
  • Open Reading Frames
  • Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Spectrophotometry
  • Spectrophotometry, Ultraviolet


  • Bacterial Proteins
  • Cytochrome b Group
  • DNA Primers
  • Iron-Sulfur Proteins
  • Ferritins
  • bacterioferritin