Ferritins, iron uptake and storage from the bacterioferritin viewpoint

Abstract

Ferritins constitute a broad superfamily of iron storage proteins, widespread in all domains of life, in aerobic or anaerobic organisms. Ferritins isolated from bacteria may be haem-free or contain a haem. In the latter case they are called bacterioferritins. The primary function of ferritins inside cells is to store iron in the ferric form. A secondary function may be detoxification of iron or protection against O(2) and its radical products. Indeed, for bacterioferritins this is likely to be their primary function. Ferritins and bacteroferritins have essentially the same architecture, assembling in a 24mer cluster to form a hollow, roughly spherical construction. In this review, special emphasis is given to the structure of the ferroxidase centres with native iron-containing sites, since oxidation of ferrous iron by molecular oxygen takes place in these sites. Although present in other ferritins, a specific entry route for iron, coupled with the ferroxidase reaction, has been proposed and described in some structural studies. Electrostatic calculations on a few selected proteins indicate further ion channels assumed to be an entry route in the later mineralization processes of core formation.

Fig. 1. (A) Overall view of the Dd Bfr [Protein Data Bank (PDB) access code 1nfv]. Different colours represent the 12 dimers. In (B), the mask (in red), highlighting the inner core, was calculated with the program MAMA (Kleywegt and Jones, 1999) with a 3 Å radius around all atoms.

Fig. 2. Two subunits from the Dd Bfr forming one homodimer. The location of the Fe-coproporphyrin III haem in the 2-fold interface is shown. The red spheres represent the di-iron ferroxidase centres in each of the monomers.

Fig. 3. The di-iron centre in Dd Bfr, showing the residues coordinating as terminal or bridging ligands. Red dashed lines indicate bonds, while black dashed lines indicate distances >2.60 Å. The distance between the iron atoms in the as ‘isolated’ structure is 3.71 Å (PDB 1nfv), while in the reduced structure it is 3.99 Å (PDB 1nf4). (A) View of the centre in the ‘as isolated’ structure with the unequivocally identified ligands. (B) View of the site in the ‘cycled’ oxidized structure (PDB 1nf6). Fe1 is strongly depleted in this structure with an occupancy between 0 and 30% in the various crystallographic independent subunits.

Fig. 4. Diagrams of Dd Bfr (A; PDB 1nfv) and human H chain ferritin subunits (B; PDB code 1fha) showing all the tyrosine and phenylalanine residues that form a possible electron transfer path from the 3-fold channel to the internal cavity of the molecule through the di-iron site.

Fig. 5. The molecular surface of the Dd Bfr homodimer (PDB 1nfv), viewed from inside the molecule, near the di-iron centre. The iron atoms are represented as green spheres with 1.30 Å radius, and the residues that coordinate the iron atoms are drawn as sticks. Asp55 and Glu131, which form hydrogen bonds with iron-coordinating residues His135 and His59, respectively, are also drawn as sticks. Glu132, which bridges the di-iron centre, is partly hidden and is not labelled for clarity. The remaining residues in the Bfr homodimer are drawn as thin white lines. The outside surface of the homodimer is near the top of the figures, while the interior surface is near the bottom. A pocket in the external surface is clearly visible in all views. This pocket is larger in the ‘native’ (not shown) and reduced Bfr (A; PDB 1nf4) than in the oxidized ‘cycled’ structure (B; PDB 1nf6). (C) The hypothetical side-chain motions of His59, Phe63 and Glu131 (drawn in yellow) in the oxidized Bfr structure, which form a channel that allows access from the depleted iron site to the inside of the Bfr 24mer. A small pocket in the internal surface can be seen in (A) and (B) just below His59, which may be the precursor of this hypothetical access channel. In (A–C), the molecular surfaces were calculated with a 1.35 Å probe radius. In (D), the molecular surface was calculated with a 0.8 Å probe radius, and a channel that may allow a Fe²⁺ ion (radius 0.76 Å) to cross the protein shell is visible. The molecular surfaces were calculated with program MSMS (Sanner et al., 1996) and the figures were prepared with DINO (Philippsen, 2002).

Fig. 6. Electrostatic potential surfaces of Dd Bfr (A and B; PDB 1nfv), Ec Bfr (C and D; PDB 1bcf) and Ec ferritin (E and F; PDB 1eum). (A, C and E) Views down a 4-fold axis (non-crystallographic). (B, D and F) Views down a 3-fold axis (crystallographic for Dd Bfr, non-crystallographic for Ec Bfr and Ec ferritin). The molecular surfaces were calculated with MSMS (Sanner et al., 1996) using a probe radius of 1.4 Å (1.2 Å for Ec Bfr). The electrostatic potentials were calculated with MEAD (Bashford, 1997) using protein and external dielectric constants of 4 and 80, respectively, a temperature of 300 K and an ionic strength of 0.1 M; for Dd Bfr, +3 charges were assumed for all the iron atoms; for Ec Bfr, +2 charges were assumed for the manganese atoms and +3 for the haem iron atoms; for Ec ferritin, no metal ions were included in the calculations as none is present in the deposited coordinates. FP, ferroxidase pore; MC, major channel; NC2, 2-fold non-crystallographic axis.

Fig. 7. Electrostatic potential surfaces of human H chain ferritin (A and B; PDB 2fha) and native horse spleen ferritin (C and D; PDB 1ier). (A and C) Views down a crystallographic 4-fold axis. (B and D) Views down a 3-fold crystallographic axis. The molecular surfaces were calculated with MSMS (Sanner et al., 1996) using a probe radius of 1.4 Å. The electrostatic potentials were calculated with MEAD (Bashford, 1997) using protein and external dielectric constants of 4 and 80, respectively, a temperature of 300 K and an ionic strength of 0.1 M; none of the metal ions present in the deposited coordinates (Ca ²⁺ for 2fha, Cd ²⁺ for 1ier) was included in the calculations.