Site-directed replacement of the coaxial heme ligands of bacterioferritin generates heme-free variants

The bacterioferritin (BFR) of Escherichia coli is a heine-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met⁵² variants were devoid of heme, whereas the Met³¹ and Met⁸⁶ variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heine-free Met⁵² variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wildtype protein. The iron contents of the Met⁵² variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heine is unnecessary for BFR assembly and iron uptake.