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.

Activity and Transcriptional Regulation of Bacterial Protein-Like Glycerol-3-Phosphate Dehydrogenase of the Haloarchaea in Haloferax volcanii

“…The GlpA2 homologs of other haloarchaea are missing the BFD-like domain. Although the physiological role of BFD domains remains unclear, this protein may serve as a general redox enzyme and/or a regulatory component of iron storage and mobilization (15). Thus, based on its genome sequence, H. volcanii harbors two separate glpABC operons that may encode functional G3PDH complexes.…”

Iron and Fur Regulation in Vibrio cholerae and the Role of Fur in Virulence

“…Bfr is a ferritin-like protein containing both heme and iron, and Bfd is believed to be a ferredoxin that participates in either the loading or removal of iron from Bfr (21,60). In E. coli (43,45,60,68) and Salmonella (5), bfd is upregulated under low-iron conditions, whereas bfr is repressed.…”

A New Role for Heme, Facilitating Release of Iron from the Bacterioferritin Iron Biomineral

“…An important observation in this context is that GSH, an abundant cytoplasmic reductant, was unable to stimulate significant iron release from E. coli BFR in vitro. Potentially important in this context is the bacterioferritin-associated ferredoxin (Bfd) (1,53,54). Expression of bfd is up-regulated under low iron, leading to the proposal that it is involved in iron release from BFR, and it has been shown previously that Bfd binds BFR specifically (47,54).…”

“…Potentially important in this context is the bacterioferritin-associated ferredoxin (Bfd) (1,53,54). Expression of bfd is up-regulated under low iron, leading to the proposal that it is involved in iron release from BFR, and it has been shown previously that Bfd binds BFR specifically (47,54). However, it is currently uncertain as to what form of the protein, the [2Fe-2S] or apo-form, is important in iron release.…”