Ferritin spectroscopy

Iron oxidized by ferritin must be at or near the outer surface of the apoferritin molecule, since iron appears to be exchanged between ferritin molecules, as shown by Mossbauer spectroscopy (Bauminger et ah, 1991a,b) and by the observation that iron oxidized by ferritin can be taken up directly by apotransferrin (Bakker and Boyer, 1986 Jin and Crichton, 1987). 

On the basis of a number of physico-chemical methods (Mossbauer spectroscopy, electron diffraction, EXAFS) the iron cores of naturally occurring haemosiderins isolated from various iron-loaded animals and man (horse, reindeer, birds and human old age) were consistently shown to have ferrihydrite-like iron cores similar to those of ferritin (Ward et ah, 1992, 2000). In marked contrast, in the tissues of patients with two pathogenic iron-loading syndromes, genetic haemochromatosis and thalassaemia, the haemosiderins isolated had predominantly amorphous ferric oxide and goethite cores, respectively (Dickson etah, 1988 Mann etah, 1988 ... 

Blnuclear Iron has been observed on ferritin coats, using EPR spectroscopy, by saturating the protein with Fe(II) (12 Fe/... 

Harrison, P.M. Fischbach, F.A. Hoy, T.G. Haggis, G.H. (1967) Ferric oxyhydroxide core of ferritin. Nature 216 1188-1190 Harvey, D.T. Linton, R.W (1981) Chemical characterization of hydrous ferric oxides by X-ray photoelectron spectroscopy. Anal. 

The major iron storage protein, ferritin, has been extensively studied (48) and shown to consist of a hollow, spherical proteinaceous shell surrounding an iron(III) oxide core. The other iron storage protein, hemosiderin, has received rather less attention, but, for normal hemosiderin, techniques such as Fe Mossbauer spectroscopy and electron diffraction (49) indicate the presence of a smaller ferritin-like core. 

In vitro a crystalline iron core can be laid down in apoferritin by the addition of an oxidant, such as O2, to an aqueous solution of a ferrous salt and apoferritin (32, 132, 140). The reconstituted core of horse ferritin prepared in the absence of phosphate and with O2 as oxidant is very similar to the native core in terms of its size and Mossbauer properties (85). Electron microscopy, however, reveals that it is less well ordered. Reconstitution in the presence of phosphate leads to smaller cores. Reconstituted A. vinelandii cores in the absence of phosphate were more ordered than were the native cores, and clearly contained ferrihydrite particles and, in some cases, crystal domains (85). Thus the nature of the core is not determined solely by the protein coat the conditions of core formation are also important. This is also indicated by Mossbauer spectroscopy studies of P. aeruginosa cells grown under conditions different than those employed for the large-scale pu-... 

As we stress in the following section, Fe + does not simply exit from the core of ferritin. Watt et al. (144) have completely reduced the core of horse ferritin and shown that it has a pH-dependent redox potential corresponding to 2H+ taken up by the core for each Fe + reduced to Fe ". In this study, anaerobic gel-filtration columns were used to separate reduced and partially reduced ferritin from contaminating small ions, and Mossbauer spectroscopy was employed to determine the extent of core reduction. Very little of the core iron was lost during the preparation, consistent with the low yields of Prussian blue formed by the addition of [FefCNle] to reduced ferritin (73, 146). Partially reduced ferritin cores have Mossbauer spectra indicative (50) of an iron... 

Electron microscopy and Mossbauer spectroscopy show (39) that the iron in hemosiderin is in the form of mineral phases, much like the iron core of ferritin. However, hemosiderin is insoluble in water at pH 7 and thus has not been chemically characterized to the same extent as ferritin. Nevertheless, the available evidence favors the formation of hemosiderin from the degradation and aggregation of ferritin (3,148). 

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). 

X-ray scattering, Mossbauer spectroscopy, infrared spectroscopy and magnetic susceptibility studies on the ferric hydroxynitrate polymer (approximate composition Fe4C>3(OH)4 (NC>3)2 1.5 H2O) shows that it is closely similar in those properties to the core of ferritin (180). It was also reported (72) that ferritin prepared from Fe2+ and apoferritin under oxidising conditions had a different morphology from native ferritin the iron micelle had a diameter of 48 A instead of 70 A and the overall diameter was 102 A instead of 120 A. 

Iron Core Only a small fraction of the iron atoms in ferritin bind directly to the protein. The core contains the bulk of the iron in a polynuclear aggregate with properties similar to ferrihydrite, a mineral found in nature and formed experimentally by heating neutral aqueous solutions of Fe(III)(N03)3. X-ray diffraction data from ferritin cores are best fit by a model with hexagonal close-packed layers of oxygen that are interrupted by irregularly incomplete layers of octahedrally coordinated Fe(III) atoms. The octahedral coordination is confirmed by Mossbauer spectroscopy and by EXAFS, which also shows that the average Fe(III) atom is surrounded by six oxygen atoms at a distance of 1.95 A and six iron atoms at distances of 3.0 to 3.3 A. 

Until recently, all ferritin cores were thought to be microcrystalline and to be the same. However, x-ray absorption spectroscopy, Mossbauer spectroscopy, and high-resolution electron microscopy of ferritin from different sources have revealed variations in the degree of structural and magnetic ordering and/or the level of hydration. Structural differences in the iron core have been associated with variations in the anions present, e.g., phosphate or sulfate, and with the electrochemical properties of iron. Anion concentrations in turn could reflect both the solvent composition and the properties of the protein coat. To understand iron storage, we need to define in more detail the relationship of the ferritin protein coat and the environment to the redox properties of iron in the ferritin core. 

The initial complexes of Fe(III) and (apo)-ferritin differ in spectral properties and rate of formation. H-type ferritins form transient blue or piiik Fe(III)-species (absorbance maximum over the range 650-550 nm) with different kinetics [40, 42, 43] and Fe(III)-oxy species which absorb over the range 350-450 nm [42, 43], L-type ferritins form only the Fe(III)-oxo species, which are indistinguishable from the mineral, preventing the measurement of decay rates by UV-visible spectroscopy. 

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