Structural studies of bacterioferritin B from Pseudomonas aeruginosa suggest a gating mechanism for iron uptake via the ferroxidase center

Abstract

The structure of recombinant Pseudomonas aeruginosa bacterioferritin B (Pa BfrB) has been determined from crystals grown from protein devoid of core mineral iron (as-isolated) and from protein mineralized with approximately 600 iron atoms (mineralized). Structures were also obtained from crystals grown from mineralized BfrB after they had been soaked in an FeSO(4) solution (Fe soak) and in separate experiments after they had been soaked in an FeSO(4) solution followed by a soak in a crystallization solution (double soak). Although the structures consist of a typical bacterioferritin fold comprised of a nearly spherical 24-mer assembly that binds 12 heme molecules, comparison of microenvironments observed in the distinct structures provided interesting insights. The ferroxidase center in the as-isolated, mineralized, and double-soak structures is empty. The ferroxidase ligands (except His130) are poised to bind iron with minimal conformational changes. The His130 side chain, on the other hand, must rotate toward the ferroxidase center to coordinate iron. In comparison, the structure obtained from crystals soaked in an FeSO(4) solution displays a fully occupied ferroxidase center and iron bound to the internal, Fe((in)), and external, Fe((out)), surfaces of Pa BfrB. The conformation of His130 in this structure is rotated toward the ferroxidase center and coordinates an iron ion. The structures also revealed a pore on the surface of Pa BfrB that likely serves as a port of entry for Fe(2+) to the ferroxidase center. On its opposite end, the pore is capped by the side chain of His130 when it adopts its "gate-closed" conformation that enables coordination to a ferroxidase iron. A change to its "gate-open", noncoordinative conformation creates a path for the translocation of iron from the ferroxidase center to the interior cavity. These structural observations, together with findings obtained from iron incorporation measurements in solution, suggest that the ferroxidase pore is the dominant entry route for the uptake of iron by Pa BfrB. These findings, which are clearly distinct from those made with Escherichia coli Bfr [Crow, A. C., Lawson, T. L., Lewin, A., Moore, G. R., and Le Brun, N. E. (2009) J. Am. Chem. Soc. 131, 6808-6813], indicate that not all bacterioferritins operate in the same manner.

Figure 1

Schematic representation of: (A) The symmetrical ferroxidase center typical of bacterioferritin. Bridging glutamates are Glu51 and Glu127 and the capping residue pairs are Glu18/His54 and Glu94/His130. (B) The ferroxidase center of human H-chain ferritin adapted from the crystal structure of the Tb³⁺ derivative (43).

Figure 2

Structure of Pa-BfrB viewed along four-fold axis (left) and along a non-crystallographic 3-fold axis. The green spheres represent potassium in each of the four-fold pores; the heme molecules are shown in gold.

Figure 3

The ferroxidase center of mineralized Pa BfrB is devoid of iron. The side chains furnishing the ligands are poised to bind iron with minimal rearrangement, with the exception of His 130 which is rotated away from the ferroxidase center.

Figure 4

Reconstitution of core iron mineral in as isolated Pa BfrB with aliquots delivering 50 Fe²⁺ ions/Pa BfrB to protein dissolved in 100 mM potassium phosphate buffer, pH 7.6 at 25 °C. The spectra, which correspond to the addition of 0, 50, 100, 150, 200, 300, 400, 500 and 600 Fe²⁺ ions, were obtained 10 min after the corresponding aliquot was added. It is noteworthy that colloidal ferric hydrous oxide was not formed, as indicated by the conservation of baseline in the family of spectra. Time-dependent changes in intensity at 320 nm were used to monitor the kinetic experiments summarized in Figure 11.

Figure 5

Views of a single subunit of Fe soaked Pa BfrB showing the iron atoms (gold spheres) located on the outer surface of the protein Fe_out, the inner surface Fe_in, and the ferroxidase iron atoms located in the center of the helix bundle.

Figure 6

Ferroxidase center of Fe soaked Pa BfrB. (A) Coordination of Fe ions at the ferroxidase center of Pa BfrB. (B) Overlay of the mineralized (green) and Fe soaked (magenta) Pa BfrB structures showing their ferroxidase centers. All residues adopt similar conformations with the exception of His130, which in the Fe-soaked structure is rotated toward the ferroxidase center, as indicated by the arrow, to facilitate binding of Fe₂. (C) His130 from the Fe soaked structure (magenta) in its coordinative and non-coordinative conformations; the latter is nearly superimposed with the His130 side chain of the mineralized structure shown in green. The 2Fo-Fc map (grey mesh) is contoured at 1σ and the Fo-Fc omit map (blue mesh) at 3σ. The Fe₂ atom is represented as an orange sphere.

Figure 7

Stereo view of the ferroxidase pore viewed from the protein exterior. The semi-transparent surface representation (magenta) is constituted by residues lining the pore wall (Asn17, Ile20, Leu93, Lys93 and Ala97). (A) The “gate-open” conformation of the Asp50 and His130 side chains (observed in the as isolated and mineralized Pa BfrB structures) allows a nearly unobstructed view of the interior cavity through the pore. (B) The “gate-closed” conformation of Asp50 and His130 (observed in the Fe-soaked structure) obstruct the bottom of the pore and poise H130 to coordinate Fe₂. The ferroxidase iron is not shown for clarity. (C) View identical as in (B) but showing the ferroxidase iron ions as orange spheres. The side chain is His130 is below Fe₂, which is located at the bottom of the pore. Fe₁ in the interior can be seen through the semi-transparent surface.

Figure 8

(A) Stereo view of a three-fold channel in the mineralized Pa BfrB structure, viewed from the interior cavity. The top of the pore is capped by the side chains of Glu109 and Asn117 (O is in red and N in blue). Below the pore becomes wider and is lined by the side chains of Lys121 and Asp118, which also undergo stabilizing electrostatic interactions. (B) Identical view of a three-fold channel in the Fe-soaked structure. The top of the pore is still capped by the interactions of Glu109 and Asn117. The interior however shows the presence of sulfate (magenta) and disorder in the side chains of Lys21. (C) View of a four fold pore where a potassium ion (yellow sphere) is coordinated by Gln151 and Asn148. The potassium ion in the four fold pore is present in as isolated, mineralized, Fe-soaked and double-soaked structures.

Figure 9

(A) A four fold-pore and neighboring B pores in the Fe-soaked Pa BfrB structure viewed from the interior cavity. The iron and potassium ions are represented by orange and green spheres, respectively. (B) An identical view in CPK rendering, which facilitates visualization of the B pores and of the four Fe_(in) ions surrounding the four-fold pore. (C) Zoomed-in view illustrating side chains that may form possible routes for moving iron ions from the ferroxidase pore, four-fold pore or B pores to their Fe_(in) position.

Figure 10

Stereo view of a single subunit showing the location of Fe_(out) on the external surface of the Fe-soaked structure, where it is coordinated by His10, His107 and three water molecules (the latter are not shown here for clarity, but can be seen in Figure S3). The view also shows the side chains of residues that may be involved in transporting the Fe_(out) to the ferroxidase pore where a disruption of the salt bridge between D100 and K96 facilitates the entry of iron to the pore. Iron ions are in orange, nitrogen atoms are in blue, oxygen atoms in red and residues lining the external outermost layer of the pore are in magenta and in CPK rendering.

Figure 11

Iron uptake in solution: Change in ΔA₃₂₀ upon subsequent additions of Fe²⁺ aliquots (50 Fe²⁺ ions per BfrB molecule) to a solution of as isolated Pa BfrB (0.8 μM) in 100 mM MES buffer, 100 mM KCl, pH 6.5. The uptake of iron was monitored with the aid of a conventional diode array spectrophotometer by acquiring spectra before the addition of an iron aliquot and every 30 seconds for 2 minutes; thereafter spectra were acquired every one minute for eight additional minutes. The responses illustrate a rapid rise in A₃₂₀ which rapidly reaches a steady value after the addition of each aliquot, suggesting that the ferroxidase center is completely vacated at the end of each plateau.

Figure 12

(A) Progress curves obtained with the aid of a stopped flow apparatus after the addition of 30, 50, 100, 200 and 300 Fe²⁺ ions per Pa BfrB molecule in 100 mM MES buffer, 100 mM KCl, pH 6.5 and 30 °C. Protein concentration is 1.0 μM (after mixing) and the path length is 1.0 cm. (B) Progress curves corresponding to the addition of 50 and 100 Fe²⁺ ions in (A) are shown to more clearly illustrate the decrease in absorbance that follows the fast phase. (C) A plot of the amplitude obtained at the end of 200 s for the progress curves in (A). (D) A plot of the amplitude of the fast phase as a function of Fe²⁺ load; the amplitudes were obtained from fitting the first 5 s of the progress curves in (A), obtained after the addition of 30, 50 and 100 Fe²⁺ ions, to a mono exponential expression.