title: A [2Fe-2S] protein encoded by an open reading frame upstream of the Escherichia coli bacterioferritin gene.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Georgia, Athens 30602, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: Garg
authors.forename: C J
authors.initials: CJ
authors.lastname: Vargo
authors.forename: X
authors.initials: X
authors.lastname: Cui
authors.forename: D M
authors.initials: DM
authors.lastname: Kurtz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005293:Ferritins / Q000737:chemistry / Q000235:genetics*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D005293:Ferritins; C018032:bacterioferritin