A [2Fe-2S] Protein Encoded by an Open Reading Frame Upstream of the Escherichia coli Bacterioferritin Gene†

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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.