Overproduction, purification and characterization of the bacterioferritin of Escherichia coli and a C‐terminally extended variant

Abstract

The bacterioferritin (BFR) of Escherichia coli is an iron-sequestering haemoprotein composed of 24 identical polypeptide chains forming an approximately spherical protein shell with a central iron-storage cavity. BFR and BFR-A, a variant with a 14-residue C-terminal extension, have been amplified (120-fold and 50-fold, respectively), purified by a new procedure and characterized. The overproduced BFR exhibited properties similar to those of natural BFR, but the iron content (25-75 non-haem Fe atoms/molecule) was 13-39-fold lower. Two major assembly states of BFR were detected, a 24-subunit protein (tetracosamer) and a novel haem-containing subunit dimer. BFR-L subunits assembled into tetracosamers having the same external-surface properties as BFR, presumably because their C-terminal extensions project into and occupy about 60% of the central cavity.As a result, BFR-A failed to take up iron under conditions that allowed incorporation into BFR in vitro. The haem content of BFR-A (1-2 haemshetracosamer) was lower than that of BFR (3.5-10.5 haems/tetracosamer) and this, together with a difference in the visible spectra of the two haemoproteins, suggested that the C-terminal extensions in BFR-A perturb the haem-binding pockets. A subunit dimer form of BFR-A was not detected. A combination of Mossbauer spectroscopy and electron diffraction showed that the BFR loaded with iron in vitro has a ferrihydrite-like iron core, whereas the in-vivo loaded protein has an amorphous core.

Structure of a Monoclinic Crystal Form of Cytochrome b1 (Bacterioferritin) from E. coli

“…This is especially significant since it has been recently demonstrated that a solution of EcBFR contains, even if in a minute quantity, dimers as well as the 24-mer protein (Andrews, Smith, Hawkins, Williams & Harrison, 1993). It has to be recognized that the molecule of BFR that we report is very surprising and raises the question of how such a water-soluble molecule can be attached to the membrane of the cell and yet sediment with membranes on cell disruption (Keilin & Harpley, 1941;Deeb & Hager, 1964).…”

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

“…Based on its similarity to other ferritins and its importance in the B. fragilis oxidative stress response, we presume that the dodecameric assembly is physiologically relevant. Importantly, recent literature for several other members of the ferritin superfamily also indicates easy dissociation into dimers or monomers in vitro (4,8,12,14,20,30). One possible explanation for BfDPSL is that assembly might be facilitated by other proteins in vivo.…”

σ S -Dependent Gene Expression at the Onset of Stationary Phase in Escherichia coli : Function of σ S -Dependent Genes and Identification of Their Promoter Sequences

“…Iron is an essential cofactor of many enzymes, and bacteria have evolved a range of strategies to acquire and store iron, which is often not bioavailable in the environment. E. coli possesses two main iron storage proteins: bacterioferritin and ferritin (4). Bacterioferritin, whose precise function is still unclear (1), is induced during slow growth or at the transition to stationary phase, consistent with its regulation by rpoS.…”