Protein-Template-Driven Formation of Polynuclear Iron Species

Abstract

Ferritins are iron-storage proteins capable of holding up to 4500 Fe(3+) ions within a single water-soluble protein shell made from 24 polypeptide chains. The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dimers of polypeptide chains being their stable forms. The aerobic addition to these of up to 8-10 or 14-20 Fe(2+) ions per dimer, respectively, results in the oxidation of the added Fe(2+) to Fe(3+). Gel permeation chromatography and sedimentation equilibrium studies confirm that the Fe(3+) ions are associated with the polypeptide dimer, and the lack of intense EPR signals from magnetically isolated Fe(3+) ions confirms the formation of one or more antiferromagnetically coupled clusters of Fe(3+) ions. The effect of Fe(3+) chelators on iron-loaded subunit dimers is to remove the Fe(3+) from the protein, but to do so slowly, consistent with it not being merely adventitiously associated with protein. These data provide experimental support for the presence of nucleation centers for the mineral cores in bacterioferritins and indicate that these proteins are not simply acting as vessels in which hydrolysis of Fe(3+) occurs independent from the protein surface. From analyses of X-ray structures and amino acid sequence comparisons, possible nucleation sites are identified.

Structural and Mechanistic Studies of a Stabilized Subunit Dimer Variant of Escherichia coli Bacterioferritin Identify Residues Required for Core Formation

“…Notably, one ethylene glycol molecule is located in an area of the protein related to the ferroxidase pore that has been identified in the structure of bacterioferritin from D. desulfuricans (8), and the other is located near the entrance at the inner surface. Electron density representing the presence of a metal ion is observed at one location of each dinuclear iron site in a position that is consistent with coordination by Glu 18 , His 54 , Glu 51 , and Glu 127 in a tetrahedral geometry. This density was modeled as a Zn 2ϩ ion, and metal ligand bonds were not restrained.…”

“…Each monomer in this minimal functional unit can form a dinuclear iron center that catalyzes the formation of a minimal iron core comprised of four to six iron atoms before precipitating (18,19). The overall kinetics of Fe 2ϩ oxidation observed on addition of Fe 2ϩ to this variant are similar to those observed for wild-type BFR but have not been reported in detail.…”

“…Protein Expression and Purification-The E128R/E135R assembly variant of E. coli BFR was expressed in E. coli MAK96 (BL21 ⌬BFR) (19) transformed with the pALN18 plasmid (18). Expression was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside when cultures achieved OD 600 ϭ 1.0, and the cells were harvested after growth for 10 h at 37°C with shaking.…”

“…Coordination of these iron atoms involves Glu 51 and Glu 127 as bridging ligands for both irons, Glu 18 and His 54 as ligands for FE1, and Glu 94 and His 130 as ligands for FE2. Previous studies of E. coli BFR have demonstrated that the ferroxidase center is essential for core formation and that core formation involves at least three kinetically distinguishable phases (11,17 ) with amino acid residues on the inner surface of the ferritin oligomer, as part of a complex and poorly defined process known as nucleation.…”

“…An assembly variant of E. coli BFR (E128R/E135R) has been shown previously to form stable subunit dimers that bind one equivalent of protoheme IX and not to form higher order oligomers (18). Each monomer in this minimal functional unit can form a dinuclear iron center that catalyzes the formation of a minimal iron core comprised of four to six iron atoms before precipitating (18,19).…”