A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral

Abstract

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.

FIGURE 1.

Iron release from E. coli BFR followed by a dithionite-ferrozine assay. A, plots of ΔA_{562 nm} as a function of time following the addition of varying concentrations (as indicated) of sodium dithionite to BFR (0.05 μm) containing ∼1200 irons/protein in MBS, 1 mm ferrozine, pH 7, at 25 °C. B, apparent rate constants, obtained from fitting the release data in A (and from similar experiments) as described under “Experimental Procedures,” plotted as a function of dithionite concentration. The left ordinate corresponds to data at pH 6 (see supplemental Fig. S2), and the right ordinate corresponds to data at pH 7. C, plots of the concentration of iron detected as the [Fe(II)(ferrozine)₃]⁴⁻ complex at the end point of each release reaction as a function of dithionite concentration. Data in B and C and in other figures are presented as averages (n ≥ 3) ± S.D. (error bars).

FIGURE 2.

Iron release from E. coli BFR followed by a dithionite/FMN-ferrozine assay. A, plots of ΔA_{562 nm} as a function of time following the addition of varying concentrations of FMN (as indicated) together with a fixed concentration of sodium dithionite (100 μm) to BFR (0.05 μm) containing ∼1200 irons/protein in MBS, 1 mm ferrozine, pH 7, at 15 °C. B, apparent rate constants at pH 6 (circles) and pH 7 (squares), obtained from fitting the data in A (and similar experiments), are plotted as a function of FMN concentration. C, the concentration of iron detected as the [Fe(II)(ferrozine)₃]⁴⁻ complex at the end point of each release reaction as a function of FMN concentration. Error bars, S.D.

FIGURE 3.

The effect of heme on iron release from E. coli BFR followed by a dithionite-ferrozine assay. A, ribbon diagram of a BFR subunit dimer generated from Protein Data Bank entry 1BCF (22) using PyMOL (57). A heme molecule (in a stick representation) is bound at the monomer-monomer interface, coordinated by Met⁵² from each subunit. The dinuclear ferroxidase center, outer, and inner protein surfaces are also indicated. B, plots of ΔA_{562 nm} as a function of time following the addition of 200 μm sodium dithionite to either wild-type BFR containing 5 hemes/24-mer or to heme-free M52H BFR. Both BFRs (0.05 μm) contained ∼1200 irons/protein and were in MBS, 1 mm ferrozine, pH 7. The release reaction was conducted at 25 °C. C, apparent rate constants, obtained from fitting the data in A (and similar experiments in which dithionite concentration was varied), plotted as a function of dithionite concentration. D, the concentration of iron detected as the [Fe(II)(ferrozine)₃]⁴⁻ complex at the end point of each release reaction as a function of dithionite concentration. Error bars, S.D.

FIGURE 4.

The effect of heme on iron release from E. coli BFR followed by a dithionite/FMN-ferrozine assay. A, plots of ΔA_{562 nm} as a function of time following the addition of 100 μm sodium dithionite and 75 μm FMN to either wild-type BFR containing 5 hemes/24-mer or to heme-free M52H BFR. Both BFRs (0.05 μm) contained ∼1200 irons/protein in MBS, 1 mm ferrozine, pH 7. The release reaction was conducted at 15 °C. B, apparent rate constants, obtained from fitting the data in A (and similar experiments in which FMN concentration was varied), plotted as a function of FMN concentration. C, the concentration of iron detected as the [Fe(II)(ferrozine)₃]⁴⁻ complex at the end point of each release reaction as a function of FMN concentration. Error bars, S.D.

FIGURE 5.

Iron reduction and release from BFR in the absence of a high affinity Fe²⁺ chelator. A, plot of ΔA_{562 nm} as a function of time following the addition of sodium dithionite (200 μm) to BFR (0.1 μm) containing ∼1200 irons/protein in MBS, pH 7, at 25 °C. Ferrozine (1 mm) was added at the indicated time following the addition of reductant, and the instantaneous observed jump in A_{562 nm} was taken to represent immediately available, released Fe²⁺. A fit of the data to a single exponential function is drawn in. B, plots of relative protein and iron concentrations following gel filtration separation of BFR and Fe²⁺ after mineral core reduction by dithionite (400 μm). BFR (0.1 μm) containing ∼1200 irons/protein in MBS, pH 7. C, as in B, but reduction was performed in the presence of ferrozine (1 mm). D, plots of ΔA_{400 nm} as a function of time following the addition of sodium dithionite (200 μm) to BFR (0.25 μm) containing ∼1200 irons/protein in MBS at 25 °C and pH 6 (circles) and pH 7 (squares). Note that full reduction of the heme present in the sample would have resulted in a decrease in A_{400 nm} of <0.005; therefore, the vast majority of the observed absorbance decrease resulted from the reduction of the Fe³⁺ mineral.

FIGURE 6.

The influence of heme and pH on iron release from BFR in the absence of a high affinity Fe²⁺ chelator. A and B, plots of ΔA_{562 nm} as a function of time following the addition of sodium dithionite (100 μm) and FMN (100 μm) to wild-type BFR containing 1 or 5 hemes/24-mer or to heme-free M52H BFR, as indicated. All BFRs (0.05 μm) contained ∼1200 irons/protein in MBS at 15 °C and pH 6 (A) and pH 7 (B). Ferrozine (1 mm) was added at the indicated time point after the addition of reductant, and the immediate jump in A_{562 nm} was taken to represent released Fe²⁺. Fits of the data to a single exponential function are drawn in. C, bar graph plots of rate constants for iron release (obtained from the fits) and percentage of total Fe³⁺ released as a function of heme content and pH, as indicated. Error bars, S.D.