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

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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‐λ, 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‐λ 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‐λ failed to take up iron under conditions that allowed incorporation into BFR in vitro. The haem content of BFR‐λ (1–2 haems/tetracosamer) 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‐λ perturb the haem‐binding pockets. A subunit dimer form of BFR‐λ was not detected. A combination of Mössbauer 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.