Vanessa J. Wade

Influence of site-directed modifications on the formation of iron cores in ferritin.

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.

Structure and composition of ferritin cores from pea seed (Pisum sativum)

Iron cores from native pea seed (Pisum sativum) ferritin have been analysed by electron microscopy and Mössbauer spectroscopy and shown to be amorphous. This correlates with their relatively high phosphate content (Fe: P = 2.83; 1800 Fe, 640 P atoms/molecule). Reconstituted cores obtained by adding iron (2000 Fe atoms/molecule) in the absence of phosphate to pea seed apoferritin were crystalline ferrihydrite. In vitro rates of formation of pea-seed ferritin iron cores were intermediate between those of recombinant human H-chain and horse spleen apoferritin and this may reflect the amino-acid residues of its ferroxidase and putative nucleation centres. The high phosphate content of pea-seed ferritin suggests that this molecule could be involved in both phosphorus and iron storage. The high phosphate concentration found within plastids, from which the molecules were isolated, is a possible source of the ferritin phosphate.

Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins.

Mössbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by Mössbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent Mössbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show Mössbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.

Structural specificity of haemosiderin iron cores in iron-overload diseases

Haemosiderin iron cores isolated from patients with secondary haemochromatosis have a goethite‐like (α‐FeOOH) crystal structure whereas those from patients with primary haemochromatosis are amorphous Fe (III) oxide. Haemosiderin cores isolated from normal human spleen are crystalline ferrihydrite (5Fe2O3·9H2O). The disease‐specific structures are significantly different from the ferrihydrite structure of associated ferritin cores. The results are important in understanding the biological processing of iron in pathological states and in the clinical treatment of iron‐overload diseases.

Biochemical studies of the iron cores and polypeptide shells of haemosiderin isolated from patients with primary or secondary haemochromatosis.

Haemosiderin isolated from different iron-loading syndromes, primary haemochromatosis (PHC) and secondary haemochromatosis (SHC) biochemically exhibited differences in both their iron core and peptide composition. The rate of release of iron from PHC haemosiderin to oxalate was 3-fold greater than that from SHC haemosiderin. The major peptides separated by SDS-PAGE showed a major band at Mr 20,000 for PHC haemosiderin and at Mr 15,000 for SHC haemosiderin.

Crystallochemical Control of Iron Oxide Biomineralization

The objective of this chapter is to identify the crystallochemical strategies evolved by organisms in the controlled mineralization of iron oxides. Three mineral phases, ferrihydrite (Fe2O3.nH2O), goethite (α-FeOOH) and magnetite (Fe3O4) will be discussed. Emphasis will be placed on the mechanisms by which organisms regulate the structure, morphology, size and organization of iron oxide biominerals and how these processes result in functional adaptation.

Mössbauer spectroscopy, electron microscopy and electron diffraction studies of small particle magnetic systems: Identification of disease specific haemosiderins

Mössbauer spectroscopy has shown that the iron-containing cores of the biological iron storage material haemosiderin produced under normal and various pathological conditions are significantly different in their magnetic properties. The differences have been correlated with information on the particle size, morphology, crystallinity and mineral form of the haemosiderin cores obtained by complementary electron microscopy and electron diffraction studies. These results have important implications for the use of Mössbauer spectroscopy in determining the properties of small particle magnetic systems and also considerable relevance for the improved understanding and treatment of iron overload disease.