Ferritin Mossbauer 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 ... 

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 Mossbauer effect has also been useful in the study of the chemical states of iron in intact cells of nitrogen-fixing bacteria. Mossbauer spectroscopy has also been used to study iron uptake and translocation in rice plants by Kilcoyne et al. (2000) who have collected spectra from intact root and leaf tissue of rice plants grown in anaerobic Fe(II)-enriched nutrient solutions. The spectra obtained from root tissue of plants grown in nutrient solutions typical of paddy soils arise primarily from Fe(III)-oxide components precipitated on the root cell walls. In contrast, the spectra obtained from root tissue from plants exposed to lower, toxic, pH conditions show that, in addition to Fe(III), uncomplexed Fe(II) is taken up. No evidence of Fe(II) was seen in the leaf tissue of any of the plants, where the spectra are characteristic of Fe(III) in ferritin and other complex forms. 

Using Mossbauer spectroscopy, three positions of trivalent iron with nonoverlapping ranges of A q were revealed in bacterial ferrihydrite nanoparticles, produced by the bacterium Klebsielh oxytoca in the course of biomineralization of iron salt solutions from a natural medium, after sample heating [60]. The fit of these spectra and interpretation of results seem to be doubtful, as the authors did not take into account a complicated iron core structure in bacterial ferritin and a core size variation while relating their results to merely two structural differences in ferrihydrite. 

The fact that iron(ll) was clearly detected by Mossbauer spectroscopy gives a direct evidence for the iron uptake mechanism of strategy I. As the time of the iron treatment increased, the reductive capacity was decreasing because of the increased amount of iron already taken up by the root [73]. According to the Mossbauer parameters of the Fe(ll), one can see that it formed a hexaaquo complex [52] that might be the primary hydrated product of the ferric chelate reductase enzyme, accumulated in the apoplast and not attached to any of the cell wall components. At the same time, the increase in the Fe(lll)A component, representing iron both attached to the apoplast and taken up inside the cell, could be observed. The ferritin-like component (denoted by Fe(lll)B) was absolutely not detectable, which means this duration of... 

Bauminger and Harrison reviewed studies of the process of iron core formation in human and horse spleen ferritins using Mossbauer spectroscopy. It was demonstrated that iron deposition within ferrihydrite core in human and horse spleen ferritin started with Fe(ll) oxidation. This process was associated with ferroxidase center of H-chains. Further, an Fe(lll) compound and Fe(lll) jL-oxo-bridged dimers in ferroxidase centers of H-chains were found, which were intermediate compounds in the process of iron oxyhydroxide core formation in horse spleen ferritin. The steps leading to ferrihydrite core formation in human L- and H-ferritins were also identified and transfer between ferritin molecules was established [M2]. 

A further development in the field of Mossbauer spectra fitting and analysis is expected regarding the explanation of applicability of either continuous distributions of quadrupole splitting and hyperfine field or a superposition of discrete quadrupole doublets and magnetic sextets with models of multidomain and multilayer structures of the ferritin iron core. In this case, application of Mossbauer spectroscopy with a high velocity resolution may be used because it leads to a lower instrumental error in the determination of hyperfine parameters (this allows small variations of hyperfine parameters to be distinguished) as well as to a more reliable fitting of complicated Mossbauer spectra (see reviews [32,34-39, 128]). 

The possible role of iron in Parkinson s, Alzheimer s, and progressive supranuclear palsy diseases was also studied using Mossbauer spectroscopy. Mossbauer studies demonstrated that the most of iron in the brain including three brain tissues substantia nigra, globus pallidus, and hippocampus was ferritin-like iron [ 134,135]. The amount of ferritin molecules... 

The smaller diameter of the iron cores of ferritin in brain compared to that in liver tissues fits well with the blocking temperatures measured by Mossbauer spectroscopy. The blocking temperature determined by MS for SN is about I5 K [20] compared to about 35-40 K for human liver [21 ]. Figure 16.4 presents the Mossbauer spectra obtained from SN at 20 K, I OK, and 4.1 K, from which the blocking temperature was estimated. 

The assessment of the concentration of labile iron in parkinsonian and control SN was made by atomic absorption after eliminating all molecules that could be bound to large molecules, such as ferritin, and thus were bigger than 10 kDa. The concentration of the labile iron in PD SN was found to be significantly higher than in controi (I35 lOngg versus 76 5 ngg wet tissue) [30]. It is important to emphasize that these iron concentrations are about 2000 times smaller than the total iron concentration and could not be detected by Mossbauer spectroscopy. 

It is important to stress that Mossbauer spectroscopy did not detect any divalent iron in all the samples measured. In a study using analytical transmission electron microscopy, the authors suggested that iron within the ferritin core in pathological SN and HP was present mainly as mixed ferric-ferrous iron oxides (magnetite-like or wiistite) and not as ferrihydrite that is the main mineral in control brain ferritin [31 ]. Mossbauer spectra of magnetite or wiistite differ from ferrihydrite spectra, but only ferrihydrite-like spectra were observed by MS in all pathological tissues. If other phases were present, they could be there only in minor quantities. 

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