Ferrihydrite nucleation sites

The initial stages of iron incorporation requires the ferroxidase sites of the protein. Thereafter, the inner surface of the protein shell provides a surface which supplies ligands that can partially coordinate iron but which leave some coordination spheres available for mineral phase anions, thereby enabling the biomineralization process to proceed, with formation of one or more polynuclear ferrihydrite crystallites. Iron is transferred from the ferroxidase sites to the core nucleation sites by the net reaction  

the overall reaction for iron oxidation and hydrolysis at the ferroxidase centre, followed by further hydrolysis and migration to the core nucleation sites consists of two reactions, the protein-catalysed ferroxidase reaction itself and the Fe(II) plus H202 detoxification reaction (Equations (19.7) and (19.8), respectively)  

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Why mammalian ferritin cores contain ferrihydrite-like structures rather than some other mineral phase is less easy to understand, and presumably reflects the way in which the biomineral is built up within the interior of the protein shell together with the geometry of the presumed nucleation sites. The phosphate content in the intracellular milieu can readily be invoked to explain the amorphous nature of the iron core of bacterioferritins and plants. Indeed, when the iron cores of bacterioferritins are reconstituted in the absence of phosphate, they are found to be more highly ordered than their native counterparts, and give electron diffraction lines typical of the ferrihydrite structure. Recently it has been reported that the 12 subunit ferritin-like Dps protein (Figure 19.6), discussed in Chapter 8, forms a ferrihydrite-like mineral core, which would seem to imply that deposition of ferric oxyhydroxides within a hollow protein cavity (albeit smaller) leads to the production of this particular mineral form (Su et al., 2005 Kauko et al., 2006). 

Figure 10 Four helix bundle of the L chain of ferritin showing iron oxide nucleating site (Glu61, Glu64, and Glu67). Quaternary structure of ferritin displaying mineralized ferrihydrite particle...

On the inside of the apoferritin shell the antiparallel B and D helices of the subunit pairs form a ridged surface partly filled by side chains and water molecules. Water molecules also cluster at the ends of helices within intersubunit contact regions. In horse spleen L ferritin, several metal-ion-binding sites have been found on the inside surface (11, 53) the possibility that some of these might represent iron sites leading to ferrihydrite nucleation has been referred to above and is discussed further in Section IV,C. 

The foreign species act in solution and usually retard nucleation or growth of goethite by competing with soluble Fe " species for sites on the subcritical nucleus or on the growing crystal. This mechanism is independent of the presence of ferrihydrite. 

There is no information on the site of formation of the Fe(III)-0-Fe(III) dimers that have been observed in rHF as well as in horse spleen ferritin (57,112). This may be clarified by Mossbauer spectroscopy on ferritin variants. Possible positions are the putative nucleation center or the double Tb site. In the latter case, either the dimers themselves must move or the iron core nucleus must incorporate iron atoms from the ferroxidase centers. Because ferrihydrite can be deposited both in rLF and in the rHF variant lacking ferroxidase activity, nucleation at this center is not possible for these molecules. Indeed, a specific center may not be required. However, electron micrographs of broken ferritin molecules suggest an attachment of ferrihydrite to the protein shell (113). 

Ferritin is a hollow shell containing 24 subunits that are a mixture of heavy (H) and light (L) chains, arranged in octahedral symmetry. The hollow core has a diameter of 8 nm and can hold as many as 4 500 iron atoms in the approximate form of the mineral ferrihydrite (5Fe203 9H2O). Iron(II) enters the protein through any of six pores located on the threefold symmetry axes of the octahedron. Oxidation to Fe(III) takes place at catalytic sites on the H chains. Sites on the inside of L chains appear to nucleate crystallization of ferrihydrite. 

Once the iron is inside the sphere, a site of mineral nu-cleation is located—step 2 in the biomineralization process. Although the site(s) of nucleation are not known, the walls are lined with carboxylic acids from aspartate and glutamate residues. These are the probable sites of nucleation. After nucleation, the mineral begins to form. The shape is controlled by the protein shell. Studies of the resulting mineral are consistent with octahedrally coordinated iron(III) ions joined by bridging oxide and/or hydroxide ions. A mineral termed ferrihydrite has a similar postulated structure. Because ferritin is used for iron storage agents, the supramolecular structures described in step 4 of Section III.B are not formed. 

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