Ferritin crystallinity

Addition of sufficient base to give a > 3 to a ferric solution immediately leads to precipitation of a poorly ordered, amorphous, red-brown ferric hydroxide precipitate. This synthetic precipitate resembles the mineral ferrihydrite, and also shows some similarity to the iron oxyhydroxide core of ferritin (see Chapter 6). Ferrihydrite can be considered as the least stable but most reactive form of iron(III), the group name for amorphous phases with large specific surface areas (>340 m2 /g). We will discuss the transformation of ferrihydrite into other more-crystalline products such as goethite and haematite shortly, but we begin with some remarks concerning the biological distribution and structure of ferrihydrite (Jambor and Dutrizac, 1998). 

When pea seed apoferritin is reconstituted in vitro in the absence of phosphate, the reconstituted mineral core consists of crystalline ferrihydrite (Rohrer et ah,1990 Wade et ah, 1993 Waldo et ah, 1995). Conversely, horse spleen ferritin reconstituted in the presence of phosphate produces an amorphous core (Rohrer et ah,1990 St. Pierre et ah, 1996)... 

However, ferritins isolated from the bacterium Pseudomonos aeruginosa (Mann et ah, 1986) and from the chiton Acanthopleura hirtosa (St. Pierre et ah, 1990) have iron cores of limited crystallinity, despite having P Fe ratios of around 1 40, perhaps suggesting that core crystallinity is influenced by the rate of iron deposition as well as by the composition of the medium. The way in which phosphate may influence core development is discussed below. 

Typically, mammalian ferritins can store up to 4500 atoms of iron in a water-soluble, nontoxic, bioavailable form as a hydrated ferric oxide mineral core with variable amounts of phosphate. The iron cores of mammalian ferritins are ferrihydrite-like (5Fe203 -9H20) with varying degrees of crystallinity, whereas those from bacterioferritins are amorphous due to their high phosphate content. The Fe/phosphate ratio in bacterioferritins can range from 1 1 to 1 2, while the corresponding ratio in mammalian ferritins is approximately 1 0.1. 

There is a number of synthetic substitutes for natural ferritin and the properties of these have been compared with those of ferritin. The synthetic polysaccharide iron complex (PIC), has a magnetic blocking temperature of 48K (Mohie-Eldin et al. 1994). Iron-dextran complexes are used as a substitute for ferritin in the treatment of anaemia. The iron cores of these complexes consist not of ferrihydrite, but of very poorly crystalline akaganeite with magnetic blocking temperatures of between 150 and 290 K (Muller, 1967 Knight et al. 1999) which were lowered from 55K to 35 and 25K, if prepared in the presence of 0.250 and 0.284 Al/(A1 -i- Fe), respectively (Cheng et al.2001). 

In crystalline oxides and hydroxides of iron (III) octahedral coordination is much more common than tetrahedral 43). Only in y-FegOs is a substantial fraction of the iron (1/3) in tetrahedral sites. The polymer isolated from nitrate solution is the first example of a ferric oxyhydroxide in which apparently all of the irons are tetrahedrally coordinated. From the oxyhydroxide core of ferritin, Harrison et al. 44) have interpreted X-ray and electron diffraction results in terms of a crystalline model involving close packed oxygen layers with iron randomly distributed among the eight tetrahedral and four octahedral sites in the unit cell. In view of the close similarity in Mdssbauer parameters between ferritin and the synthetic poljmier it would appear unlikely that the local environment of the iron could be very different in the two materials, whatever the degree of crystallinity. Further study of this question is needed. 

The chemistry of the iron core is most interesting, in the light of the experiments mentioned above with respect to the hydrolytic polymers of Fe (III). The molecular structure of the iron core obtained from crystalline ferritin by treatment with concentrated sodium hydroxide has recently been investigated using low angle X-ray scattering patterns (44). These authors have proposed a structure quite similar to that of Green rust II ... 

Protein subunits will partially dissociate from crystalline ferritin in dilute salt solutions to yield a non-crystallizable ferritin. The non-crystallizable ferritin, in turn, in the presence of apoferritin appears to pick up protein subunits and by action yield crystalline ferritin molecules. The scheme for this process is shown in Fig. 6. The salient feature of this scheme is the initial formation of an iron micelle from soluble iron chelates which is then stabilized by protein subimits S5uithesized by the tissue (726). Harrison and Gregory 127) have used glacial acetic acid to dissociate apoferritin completely. When the pH is adjusted to 4 in the presence of thiol compoimds, apoferritin is rapidly formed, indicating strong subunit interaction. 

The inner cavity is filled with small crystalline particles of composition [Fe(0)(0H)]g-[Fe(0)(0P03H2)], which is 57% iron and contains up to about 4500 iron atoms per ferritin molecule. While the core stores iron as Fe111, it is generally accepted that deposition and mobilization involve Fe11. Apoferritin rapidly accumulates iron in a solution containing Fe11 and 02, but Fe111... 

The concentrations of iron as simple solvated ions, Fe2+aq or Fe3+aq, are maintained at extremely low levels because of their damaging ability in the presence of oxygen and hydrogen peroxide. In transferrin, an important iron scavenger, the iron is well-protected and is not involved in redox chemistry. Also, in the major storage proteins, ferritin and haemosiderin, the iron is present in crystalline material inside the protein shell, and is well protected from reaction. 

Fe(III) particles through oxidation of Fe(n) at sites different from the intra-subunit ferroxidase centers of H-chain ferritins. These sites involve glutamic acid residues 53, 56, 57 and 60, and face the inner cavity of the molecule. Because ferritins rich in 1-chains oxidize iron more slowly than H-chain ferritins, they form iron particles of greater average size, crystallinity, and magnetic ordering. ... 

The generally accepted structure for the crystalline iron core of ferritin is the ferrihydrite structure proposed by Towe Bradley.This consists of oxygen layers with iron in octahedral sites between the layers. Ford et al and PowelF have sununarized the evidence in support of this model for ferritin, which includes electronic spectroscopic data and EXAFS measurements confirming the presence of six-coordinate Fe(III) and indicating that four-coordinate Fe(III) is present, if at aU, at low levels only. This latter point is important because the X-ray powder diffraction patterns of... 

Reconstitution experiments with apoferritins from animal and bacterial sources, whose native iron-loaded ferritins had crystalline and amorphous cores respectively, have been informative in showing that the core morphology is not determined by the protein shell. For example, Baaghil et al and Mann etalP were able to form crystalline cores in bacterioferritins, and Rohrer formed cores of iron-... 

A variety of approaches have been adopted in attempts to obtain analogs of crystalline ferritin cores but, as Powell has pointed out, uncertainty over the structure of the ferritin core itself complicates this field. The earliest synthetic approach was that of Spiro et al., who obtained citrate-coated spheres of polymeric iron hydroxide by hydrolysis of ferric citrate. Though the early characterization of these spheres indicated they might be good models for ferritin, the difficulty in obtaining reproducible samples, and the lack of structural information, has led to greater efforts being put into other approaches. 

Additionally, the L. innocua ferritin-like protein served as a template for the controlled mineralization of two cobalt oxide phases Co(0)OH and C03O4 under two reaction temperatures of 23° and 65 °C, respectively. Substantial differences in crystallinity of the cobalt mineral core was observed between the two synthetic routes. The mineralization reaction carried out at higher temperatures yielded more crystalline nanomaterials, while the low-temperature synthesis tended toward amorphous material. The high crystallinity obtained at higher temperatures is most likely due to removal of structural waters present in the protein cavity and the surpassed energy barrier of nucleation at 65 °C. ... 

The structure of ferritin cores has been studied by X-ray diffraction (47), electron diffraction (48), and high-resolution electron microscopy (49,50). The results indicate that the iron-containing cores of mammalian ferritins are crystalline with a unit cell based on a four-layer... 

The core of horse and human ferritin has been identified as crystalline ferrihydrite, 5Fe203 9H2O (49, 137), with some adventitious phosphate bound at the surface of the core. The proportion of phosphate to iron is very low. By contrast, the cores of bacterial ferritin are... 

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-... 

Ferritin source Fe atoms/ molecule Fe atoms/ phosphate" Fe atoms/ P atoms Crystallinity n 7 ord Ref"... 

In contrast to ferritin, very little work has been done on the reconstitution of BFR cores, other than the experiments mentioned above that showed that, in the absence of phosphate, crystalline ferrihydrite formed inside the protein shell. The intermediate stages in this process are unknown, but the sigmoid iron uptake behavior (25) suggests there could be a similar succession of events oxidation and nucleation on the protein shell followed by direct oxidation on the core. The influence of the heme, if any, on BFR iron core formation also awaits investigation. As mentioned above, the presence of the iron core influences the heme redox potential, but it is not known whether the presence of heme influences the redox potential of the nonheme iron. 

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