N. Dennis Chasteen

Ferritin is a widespread iron mineralizing and detoxification protein that stores iron as a hydrous ferric oxide mineral core within a shell-like structure of 4/3/2 octahedral symmetry. Iron mineralization is initiated at dinuclear ferroxidase centers inside the protein where Fe²⁺ and O₂ meet and react to form a μ-1,2-peroxodiferric intermediate that subsequently decays to form μ-oxo dimeric and oligomeric iron(III) species and ultimately the mineral core. Several types of channels penetrate the protein shell and are possible pathways for the diffusion of iron and dioxygen to the ferroxidase centers. In the present study, UV/visible and fluorescence stopped-flow spectrophotometries were used to determine the kinetics and pathways of Fe²⁺ diffusion into the protein shell, its binding at the ferroxidase center and its subsequent oxidation by O₂. Three fluorescence variants of human H-chain ferritin were prepared in which Trp34 was introduced near the ferroxidase center. They included a control variant no. 1 (W93F/Y34W), a “1-fold” channel variant no. 2 (W93F/Y34W/Y29Q) and a 3-fold channel variant no. 3 (Y34W/W93F/D131I/E134F). Anaerobic rapid mixing of Fe²⁺ with apo-variant no. 1 quenched the fluorescence of Trp34 with a rate exhibiting saturation kinetics with respect to Fe²⁺ concentration, consistent with a process involving facilitated diffusion. A half-life of ∼3 ms for this process is attributed to the time for diffusion of Fe²⁺ across the protein shell to the ferroxidase center. No fluorescence quenching was observed with the 3-fold channel variant no. 3 or when Zn²⁺ was prebound in each of the eight 3-fold channels of variant no. 1, observations indicating that the hydrophilic channels are the only avenues for rapid Fe²⁺ access to the ferroxidase center. Substitution of Tyr29 with glutamine at the entrance of the “1-fold” hydrophobic channel had no effect on the rate of Fe²⁺ oxidation to form the μ-1,2-peroxodiferric complex (t_1/2 ≈ 38 ms), a finding demonstrating that Tyr29 and, by inference, the “1-fold” channels do not facilitate O₂ transport to the ferroxidase center, contrary to predictions of DFT and molecular dynamics calculations. O₂ diffusion into ferritin occurs on a time scale that is fast relative to the millisecond kinetics of the stopped-flow experiment.

Electron paramagnetic resonance (EPR) signals at g′ = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe³⁺ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g′ = 4.3 intensity standard and for accurately quantifying the amount of Fe³⁺ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308–315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe³⁺–EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu²⁺–EDTA and aqua VO²⁺, both S = 1/2 ions, and of Fe³⁺–transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of ±3.6, ±5.3 and ±2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe³⁺–desferrioxamine of known Fe³⁺ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.

Zinc and terbium, inhibitors of iron incorporation in the ferritins, have been used for many years as probes of structure-function relationships in these proteins. Isothermal titration calorimetric and kinetic measurements of Zn(II) and Tb(III) binding and inhibition of Fe(II) oxidation were used to identify and characterize thermodynamically (n, K, ΔH°, ΔS°, and ΔG°) the functionally important binding sites for these metal ions in recombinant human H-chain, L-chain, and H-chain site-directed variant ferritins. The data reveal at least two classes of binding sites for both Zn(II) and Tb(III) in human H-chain ferritin: one strong, corresponding to binding of one metal ion in each of the eight three-fold channels, and the other weak, involving binding at the ferroxidase and nucleation sites of the protein as well as at other weak unidentified binding sites. Zn(II) and Tb(III) binding to recombinant L-chain ferritin showed similar stoichiometries for the strong binding sites within the channels, but fewer weaker binding sites when compared to the H-chain protein. The kinetics and binding data indicate that the binding of Zn(II) and Tb(III) in the three-fold channels, which is the main pathway of iron(II) entry in ferritin, blocks the access of most of the iron to the ferroxidase sites on the interior of the protein, accounting for the strong inhibition by these metal ions of the oxidative deposition of iron in ferritin.

Biomineralization of the ferritin iron core involves a complex series of events in which H₂O₂ is produced during iron oxidation by O₂ at a dinuclear centre, the ‘ferroxidase site’, located on the H-subunit of mammalian proteins. Rapid-freeze quench Mössbauer spectroscopy was used to probe the early events of iron oxidation and mineralization in recombinant human ferritin containing 24 H-subunits. The spectra reveal that a μ-1,2-peroxodiFe(III) intermediate (species P) with Mössbauer parameters δ (isomer shift) = 0.58mm/s and ΔE_Q (quadrupole splitting) = 1.07mm/s at 4.2K is formed within 50ms of mixing Fe(II) with the apoprotein. This intermediate accounts for almost all of the iron in the sample at 160ms. It subsequently decays within 10s to form a μ-oxodiFe(III)—protein complex (species D), which partially vacates the ferroxidase sites of the protein to generate Fe(III) clusters (species C) at a reaction time of 10min. The intermediate peroxodiFe(III) complex does not decay under O₂-limiting conditions, an observation suggesting inhibition of decay by unreacted Fe(II), or a possible role for O₂ in ferritin biomineralization in addition to that of direct oxidation of iron(II).

Iron deposition in the unusual 12-subunit ferritin from the bacterium Listeria innocua proceeds in three phases: a rapid first phase in which Fe²⁺ binds to the apoprotein, P^Z of charge Z, according to the postulated reaction 2Fe²⁺+P^Z → [Fe₂–P]^Z+2+2H⁺, where [Fe₂–P]^Z+2 represents a dinuclear iron(II) complex formed at each of the 12 ferroxidase centres of the protein; a second phase corresponding to oxidation of this putative complex, i.e. [Fe₂–P]^Z+2+½O₂ → [Fe₂O–P]^Z+2H⁺; and a third phase of iron(II) oxidation/mineralization, i.e. 4Fe²⁺+O₂+8H₂O → 8FeOOH_(s)+8H⁺ [where FeOOH_(s) represents the hydrous ferric oxide mineral that precipitates from the solution], which occurs when iron is added in excess of 24Fe²⁺/protein. In contrast with other ferritins, the ferroxidation reaction in L. innocua ferritin proceeds more slowly than the oxidation/mineralization reaction. Water is the final product of dioxygen reduction in the 12-subunit L. innocua ferritin (the present work) and in the 24-subunit Escherichia coli bacterioferritin, whereas H₂O₂ is produced in 24-subunit mammalian ferritins. Possible reasons for this difference are discussed.