John M. Williams

On the structure of hemosiderin and its relationship to ferritin

The atomic structures and morphology of the mineral components of ferritin and hemosiderin prepared from a single horse spleen are compared by X-ray diffraction, Mossbauer spectroscopy, and electron microscopy. The atomic structures could not be distinguished by X-ray diffraction and Mossbauer spectroscopy, although the average particle size in hemosiderin appears to be somewhat smaller than that in ferritin, as judged by X-ray diffraction and electron microscopy. The results are compatible with the conclusion of earlier studies that hemosiderin may consist largely of ferritin ironcores, which have aggregated on removal of their protein coats by proteolytic digestion. However, formation of such particles independently of ferritin is possible.

Reconstituted and native iron-cores of bacterioferritin and ferritin

The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the Az. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn2+ reduced the initial rate of Fe(II) oxidation in Az. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous. However, the blocking temperature for reconstituted Az. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that the reconstituted Az. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume (V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant (K) in such a way that the magnetic anisotropy barrier (KV), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in vivo.

Studies on haemosiderin and ferritin from iron-loaded rat liver

SummaryHaemosiderin has been isolated from siderosomes and ferritin from the cytosol of livers of rats iron-loaded by intraperitoneal injections of iron-dextran. Siderosomal haermosiderin, like ferritin, was shown by electron diffraction to contain iron mainly in the form of small particles of ferrihydrite (5Fe2O3 · 9H2O), with average particle diameter of 5.36±1.31 nm (SD), less than that of ferritin iron-cores (6.14±1.18 nm). Mössbauer spectra of both iron-storage complexes are also similar, except that the blocking temperature,TB, for haemosiderin (23 K) is lower than that of ferritin (35 K). These values are consistent with their differences in particle volumes assuming identical magnetic anisotropy constants. Measurements of P/Fe ratios by electron probe microanalysis showed the presence of phosphorus in rat liver haemosiderin, but much of it was lost on extensive dialysis. The presence of peptides reacting with anti-ferritin antisera and the similarities in the structures of their iron components are consistent with the view that rat liver haemosiderin arises by degradation of ferritin polypeptides, but its peptide pattern is different from that found in humanβ-thalassaemia haemosiderin. The blocking temperature, 35 K, for rat liver ferritin is near to that reported, 40 K, for humanβ-thalassaemia spleen ferritin. However, the haemosiderin isolated from this tissue, in contrast to that from rat liver, had aTB higher than that of ferritin. The iron availability of haemosiderins from rat liver and humanβ-thalassaemic spleen to a hydroxypyridinone chelator also differed. That from rat liver was equal to or greater, and that from human spleen was markedly less, than the iron availability from either of the associated ferritins, which were equivalent. The differences in properties of the two types of haemosiderin may reflect their origins from primary or secondary iron overload and differences in the duration of the overload.

The thickness dependence of Mössbauer absorption line areas in unpolarized and polarized absorbers

Abstract Mossbauer absorption theory is presented with particular attention to the dependence of line areas on thickness. The saturation of the line area ratios of two lines within a hyperfine-split 57 Fe spectrum is studied, and from thickness dependence measurements in both unpolarized and polarized spectra a new method of estimating recoil-free fractions is presented.

The relationship between ferritin and haemosiderin

Under conditions of iron overload haemosiderin may replace ferritin as a major iron store. Much of this haemosiderin is contained within secondary lysosomes, known as sidersomes, where it is thought to have arisen by breakdown of ferritin. We report on57Fe Mössbauer studies of ferritin and haemosiderin prepared from iron loaded rat livers and measured over a wide temperature range. Our results indicate significantly different superparamagnetic blocking temperatures consistent with the model whereby haemosiderin is derived from ferritin by lysosomal degradation.

Lability of iron at the dinuclear centres of ferritin studied by competition with four chelators

Abstract Ferritin molecules contain 24 polypeptide chains folded as four-helix bundles and arranged as a hollow shell capable of storing up to 4500 Fe(III) atoms. H chains contain ferroxidase centres which lie within the bundle, about 12 Å (1.2 nm) from the outside surface and 8 Å from the inner surface of the protein shell. Catalysis of Fe(II) oxidation precedes storage of Fe(III) as ferrihydrite, with the formation of μ-oxo-bridged Fe(III) dimers as intermediates. Factors influencing the movement of μ-oxo-bridged Fe(III) from the ferroxidase centre to the ferritin cavity are uncertain. Assistance by small chelators is one possibility. The aim of this investigation was to determine whether iron at the dinuclear centres of three ferritins (human H chain homopolymer, HuHF, the non-haem ferritin of Escherichia coli, EcFTN, and horse spleen ferritin, HoSF) is accessible to chelators. Forty-eight Fe(II) atoms/molecule were added to the apoferritins followed, 2 min later, by the addition of chelator (1,10-phenanthroline, 2,2-bipyridine, desferrioxamine or 3,4-dihydroxybenzaldehyde). Iron species were analysed by Mössbauer spectroscopy or visible absorbance. Competition between chelators and apoferritin for Fe(II) was also investigated. The main conclusions of the study are that: (1) dinuclear iron and iron in small iron-cores in HuHF and EcFTN is mobilisable by all four chelators; (2) the chelators penetrate the shell; (3) 3,4-dihydroxybenzaldehyde is the most efficient in mobilising Fe(III) but the least successful in competing for Fe(II); (4) Fe(III) is more readily released from EcFTN than from HuHF; (5) 2,2′-bipyridine aids the movement of Fe(III) from ferroxidase centre to core.

Mössbauer studies of the ultrafine antiferromagnetic cores of ferritin

The ultrafine iron cores found within the iron-storage protein ferritin are of interest to both the molecular biologist and physicist. The manner in which the protein shell (apoferritin) takes up, releases and sequesters iron is of great biological importance. Also, due to their nano-size (≤8 nm), the magnetically ordered cores are singly domained. Such particles possess interesting magnetic relaxation and dynamic properties for which Mössbauer spectroscopy is an ideal analytic tool. Horse ferritin molecules iron-loaded artificially contain ferrihydrite-like cores which behave as superparamagnets due to their nano-size. An anomalous decrease in thef-factor occurs above the blocking temperature of the superparamagnetic particles. This has been attributed to a magnetostriction effect resulting from superparamagnetic switching.

Mössbauer absorption line function for a polycrystalline quadrupole-split absorber situated in a large applied magnetic field

Abstract Including first and second order approximations for the excited state nuclear energy level shifts in 57 Fe for mixed electric quadrupole and magnetic dipole interactions, where the latter is dominant, we have solved the absorption line integral for a polycrystalline absorber situated in a relatively large applied magnetic field. Such interactions are shown to lead to a distortion and shift of the individual absorption lines. The possibility of observing experimentally the resulting theoretical line forms is also discussed.

Variable temperature 119mSn Mössbauer studies of Ph3SnSCH2CH2COOSnPh3 and Bu3SnSCH2CH2COOSnBu3

Abstract Variable temperature 119m Sn Mossbauer data are presented for two organotin compounds which each contain 4- and 5-coordinate tin sites. The absorption line areas of each site have been determined and the Debye model of solids has been used to fit the experimental data. The Debye temperature associated with each site has been determined, and compared with other data.

Iron(III) species formed during iron(II) oxidation and iron-core formation in the bacterioferritin of Escherichia coli

SummaryThis paper describes a preliminary investigation of the mechanisms of Fe(II) oxidation and storage of Fe(III) in the bacterioferritin of Escherichia coli (EcBFR). Using Mössbauer spectroscopy to examine the initial oxidation of iron by EcBFR we have confirmed that this ferritin exhibits «ferroxidase» activity and have shown that dimeric and monomeric iron species are produced as intermediates. We compare the characteristics of ferroxidase activity in EcBFR with those of human H-chain ferritin (HuHF) and discuss the different Mössbauer parameters of their dimeric iron with reference to the structures of their di-metal sites. In addition, we present preliminary findings suggesting that after an initial «burst», the rate of oxidation is greatly reduced, possibly due to blockage of the ferroxidase centre by bound iron. A new component, not found in HuHF and probably representing a small cluster of Fe(III) atoms, is reported.