Moessbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated iron(III) atoms and oxo-bridged iron(III) dimers as early intermediates

Abstract

Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room temperature before freezing). The measurements made at 4.1 and 90 K showed that for samples with n less than or equal to 40 at pH greater than or equal to 6.25 all iron was trivalent at tf = 3 min. Four different Fe(III) species were identified: solitary Fe(III) atoms giving relaxation spectra, which can be identified with the species observed before by EPR and UV difference spectroscopy; oxo-bridged dimers giving doublet spectra with large splitting, observed for the first time in ferritin; small Fe(III) clusters giving doublets of smaller splitting and larger antiferromagnetically coupled Fe(III) clusters, similar to those found previously in larger ferritin iron cores, which, for samples with n greater than or equal to 40, gave magnetically split spectra at 4.1 K. Both solitary Fe(III) and dimers diminished with time, suggesting that they are intermediates in the formation of the iron core. Two kinds of divalent iron were distinguished for n = 480, which may correspond to bound and free Fe(II).

Variation of superparamagnetic properties with iron loading in mammalian ferritin

“…This could be due to different methods of addition of ferric iotas to the core, i.e., incubation with ferric ions [20], or, anaerobic incubation with ferrous ions followed by air oxidation (present experiments). These conditions, or incubation with ferrous iron in the presence of air [23,47], could affect the distribution of ferric ions in the ferritin cores. Other factors affecting distribution might include pH, phosphate content, and ageing (i.e., loss of water and rearrangement of ions) of the core particles.…”

“…A variety of physical techniques including X-ray and electron diffraction [6,7], high resolution transmission electron microscopy [8,9], EXAFS [10], EPR [11], NMR relaxometry [12,13], magnetic measurements [14][15][16][17], and M/Sssbauer spectroscopy [10, 14,[18][19][20][21][22][23][24][25][26] have been used to study the properties of the ferric oxy-hydroxide core particles of ferritin. The particles consist of the quasicrystalline, ferric-hydrous-oxide mineral, ferrihydrite.…”

Overproduction, purification and characterization of the bacterioferritin of Escherichia coli and a C‐terminally extended variant

“…This was achieved by five successive additions (5, 10, 15, 30 and 35pI) 17.5 mM 57FeS04 (prepared according to Bauminger et al, 1989) at 10-min intervals to 530 pg BFR in 3 ml 20 mM Hepes, pH 7.0. The resulting solution was then air dried.…”

Iron core formation in horse spleen ferritin: Magnetic susceptibility, pH, and compositional studies

“…Previous Mossbauer measurements [11] of reconstituted HoSF have shown a succession of transient, monomeric, dimeric, and iron cluster species, with concentrations changing as a function of time as measured in minutes. The results reported here probably represent relatively "static" iron cores in HoSF but nevertheless suggest the presence of isolated Fe 3 + compara ble in concentration to those reported for short term measurements [11].…”

“…It has been proposed that Oz-oxidation of Fe 2 + by apofer ritin initially occurs at the ferroxidase centers, followed by migration of Fe3+ to mineral nucleation sites in the cavity [8J. Oxidation of the ferrous ions in the ferroxidase center is thought to involve formation of isolated Fe3+ [9, 10J as well as oxo dimers [10,11]. Because bacterial ferritins have only one subunit type, the concept of H subunit containing the ferroxidase center needs to be further examined for the bacterial ferritin system.…”