Structural Characterization of Bacterioferritin from <em>Blastochloris viridis</em>

<div><p>Iron storage and elimination of toxic ferrous iron are the responsibility of bacterioferritins in bacterial species. Bacterioferritins are capable of oxidizing iron using molecular oxygen and import iron ions into the large central cavity of the protein, where they are stored in a mineralized form. We isolated, crystallized bacterioferritin from the microaerophilic/anaerobic, purple non-sulfur bacterium <em>Blastochloris viridis</em> and determined its amino acid sequence and X-ray structure. The structure and sequence revealed similarity to other purple bacterial species with substantial differences in the pore regions. Static 3- and 4-fold pores do not allow the passage of iron ions even though structural dynamics may assist the iron gating. On the other hand the B-pore is open to water and larger ions in its native state. In order to study the mechanism of iron import, multiple soaking experiments were performed. Upon Fe(II) and urea treatment the ferroxidase site undergoes reorganization as seen in bacterioferritin from <em>Escherichia coli</em> and <em>Pseudomonas aeruginosa</em>. When soaking with Fe(II) only, a closely bound small molecular ligand is observed close to Fe₁ and the coordination of Glu94 to Fe₂ changes from bidentate to monodentate. DFT calculations indicate that the bound ligand is most likely a water or a hydroxide molecule representing a product complex. On the other hand the different soaking treatments did not modify the conformation of other pore regions.</p> </div

Hole2 representations of the 4 fold pore.

<p>(A) Overview of the <i>Bv</i> Bfr 4-fold pore (B) Zoom onto the residues immediately surrounding the 4-fold pore in <i>Bv</i> Bfr and (C) <i>Pa</i> Bfr 4-fold pore with a potassium ion (<i>pink</i>) modeled in the center of the pore (PDB entry 3ISF) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone.0046992-Weeratunga1" target="_blank">[8]</a>.</p

Hole2 representations of the 3 fold pore.

<p>(A) Overview of the <i>Bv</i> Bfr 3-fold pore (B) constraining residues in the <i>Bv</i> Bfr 3-fold pore (C) Superposition of <i>Bv</i> Bfr (<i>brown</i>) and <i>Ec</i> Bfr 3-fold pore (<i>grey</i>) (PDB entry 2Y3Q) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone.0046992-Antonyuk1" target="_blank">[42]</a>.</p

Interatomic distances in the different <i>Bv</i> Bfr structures compared to the DFT optimized distances from Compounds A and B from Figure S1.

<p>Interatomic distances in the different <i>Bv</i> Bfr structures compared to the DFT optimized distances from Compounds A and B from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone.0046992.s001" target="_blank">Figure S1</a>.</p

Normalized crystallographic B-factors of the pore forming residues upon “Fe-soaked” and “double soaked” treatments.

<p>Normalized crystallographic B-factors of the pore forming residues upon “Fe-soaked” and “double soaked” treatments.</p

Crystallographic data and refinement statistics.

a<p>Values in parentheses indicate statistics for the highest resolution shell.</p>b<p><i>R</i>_sym = ∑ |Io−<i>|/∑ Io×100%, where Io is the observed intensity of a reflection and <i> is the average intensity obtained from multiple observations of symmetry related reflections.</i></i></p><i><i>c<p><i>R</i>_factor =∑ ||F_obs|-|F_calc||/∑ |F_obs|×100%.</p>d<p>Average <i>B</i>-factor of amino acids shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone-0046992-g006" target="_blank">Figure 6B</a>.</p>e<p>Average <i>B</i>-factor of amino acids shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone-0046992-g007" target="_blank">Figure 7B</a>.</p></i></i

Ferroxidase site in the open and closed state.

<p>His130 from ‘as-isolated’ structure (<i>gray</i>) is rotated toward the ferroxidase center to facilitate binding of Fe₂, while in the double-soaked structure (<i>green</i>) the His130 is in its non-coordinative conformation. Hole2 channel through the ferroxidase site was generated using the double-soaked structure with both Fe₁ and Fe₂ removed.</p

DFT calculations.

<p>Stick-ball representation of ferroxidase center from DFT calculations superimposed on the Fe-soaked crystal structure. The ligand to Fe₁ is (A) a water molecule, with mixed valence and high multiplicity; (B) a hydroxide ion, with Fe₁ reduced and Fe₂ oxidized and both at high spin. The model with <i>green</i> and <i>yellow</i> carbon atoms represents the crystal structure and the DFT optimized coordinates, respectively.</p

The ferroxidase center of <i>Bv</i> Bfr in stereo view of electron density in 2mF_o-DF_c maps.

<p> (A) The structure of as-isolated, (B) Fe-soaked and (C) double-soaked <i>Bv</i> Bfr active site, respectively. The 2mF_o-DF_c map (blue) is contoured at 1σ, 2σ and 1.5σ, respectively. The dashed red lines show the coordinating bonds to the iron atoms and the dashed black lines indicate hydrogen bonds.</p

Hole2 representation of the <i>Bv</i> Bfr B-pore.

<p>The side chains of amino acid residues surrounding the constricted region of the pore are also indicated.</p