Reductive Mobilization of Iron from Intact Ferritin: Mechanisms and Physiological Implication

Abstract

Ferritins are highly conserved supramolecular protein nanostructures composed of two different subunit types, H (heavy) and L (light). The two subunits co-assemble into a 24-subunit heteropolymer, with tissue specific distributions, to form shell-like protein structures within which thousands of iron atoms are stored as a soluble inorganic ferric iron core. In-vitro (or in cell free systems), the mechanisms of iron(II) oxidation and formation of the mineral core have been extensively investigated, although it is still unclear how iron is loaded into the protein in-vivo. In contrast, there is a wide spread belief that the major pathway of iron mobilization from ferritin involves a lysosomal proteolytic degradation of ferritin, and the dissolution of the iron mineral core. However, it is still unclear whether other auxiliary iron mobilization mechanisms, involving physiological reducing agents and/or cellular reductases, contribute to the release of iron from ferritin. In vitro iron mobilization from ferritin can be achieved using different reducing agents, capable of easily reducing the ferritin iron core, to produce soluble ferrous ions that are subsequently chelated by strong iron(II)-chelating agents. Here, we review our current understanding of iron mobilization from ferritin by various reducing agents, and report on recent results from our laboratory, in support of a mechanism that involves a one-electron transfer through the protein shell to the iron mineral core. The physiological significance of the iron reductive mobilization from ferritin by the non-enzymatic FMN/NAD(P)H system is also discussed.

Keywords: chaotropes; electron transfer; ferritin; flavin nucleotide; iron mobilization; kinetics.

Figure 1

Ferritin molecule (PDB 1R03) with highlighted hydrophobic four-fold (left), and hydrophilic three-fold channels (right), that allow the transport of small molecules and ions to the inner cavity.

Figure 2

Schematic depiction of the oxidative deposition and reductive mobilization of iron in ferritin.

Figure 3

Reductive mobilization of iron from HosF containing 2250 Fe/shell. Conditions: 0.2–1 μM ferritin, 2 mM NADH, 200 μM FMN, 2 mM 2,2′-bipyridine, 2650 U/mL catalase, at pH 7.0 and 22 °C. (A) Absorbance change of [Fe(bipy)₃]²⁺ as a function of time. (B) Change of the iron(II)–bipyridine release rate versus time for different concentrations of HosF. (C,D) Reductive mobilization of iron from human recombinant heteropolymer ferritin (0.2 and 0.4 μM) loaded with 500 Fe/protein, in the presence of 2 mM NADH, 200 μM FMN, 2 mM 2,2′-bipyridine, 2650 U/mL catalase, at pH 7.0 and 28 °C. (C) Absorbance change of [Fe(bipy)₃]²⁺ as a function of time. (D) Change of the iron(II)–bipyridine release rate versus time, for different concentrations of heteropolymer ferritin. Reprinted with permission from Ref. [23].

Figure 4

Schematic of the simultaneous monitoring of the dissolved oxygen concentration (blue curve), and light absorption of Fe(II)–bipyridine complex at 530 nm (red curve), during the reductive release of iron from HosF (0.6 μM), in the presence of 2 mM NADH, 2 mM FMN, and 2 mM 2,2′-bipyridine. Reprinted with permission from Ref. [23].

Figure 5

Schematic of Fe²⁺ cations diffusion and electron transfer across the ferritin shell, followed by Fe²⁺ oxidation and deposition.

Figure 6

Rates of iron release from HosF and HuHF by the FMN-NADH system (A–D), and by FMNH₂ (E), in the absence or presence of different concentrations of chaotropes. In both experiments, the presence of urea does not affect the rates of iron mobilization. Reprinted with permission from Ref. [25].

Figure 7

Structures of specific iron(III) chelators.

Figure 8

Superoxide mediated mobilization of iron(III) cations from the ferritin iron core by BHT (or DFO) chelators.