Concerted Motions Networking Pores and Distant Ferroxidase Centers Enable Bacterioferritin Function and Iron Traffic

Abstract

X-ray crystallography, molecular dynamics (MD) simulations and biochemistry were utilized to investigate the effect of introducing hydrophobic interactions in the 4-fold (N148L and Q151L) and B-pores (D34F) of Pseudomonas aeruginosa bacterioferritin B (BfrB) on BfrB function. The structures show only local structural perturbations and confirm the anticipated hydrophobic interactions. Surprisingly, structures obtained after soaking crystals in Fe2+-containing crystallization solution revealed that although iron loads into the ferroxidase centers of the mutants, the side chains of ferroxidase ligands E51 and H130 do not reorganize to bind the iron ions, as is seen in the wt BfrB structures. Similar experiments with a double mutant (C89S/K96C) prepared to introduce changes outside the pores show competent ferroxidase centers that function akin to those in wt BfrB. MD simulations comparing wt BfrB with the D34F and N148L mutants show that the mutants exhibit significantly reduced flexibility, and reveal a network of concerted motions linking ferroxidase centers and 4-fold and B-pores, which are important for imparting ferroxidase centers in BfrB with the required flexibility to function efficiently. In agreement, the efficiency of Fe2+ oxidation and uptake of the 4-fold and B-pore mutants in solution is significantly compromised relative to wt or C89S/K96C BfrB. Finally, our structures show a large number of previously unknown iron binding sites in the interior cavity and B-pores of BfrB, which reveal in unprecedented detail conduits followed by iron and phosphate ions across the BfrB shell, as well as paths in the interior cavity that may facilitate nucleation of the iron phosphate mineral.

Inhibiting the BfrB:Bfd interaction in Pseudomonas aeruginosa causes irreversible iron accumulation in bacterioferritin and iron deficiency in the bacterial cytosol

“…These concerted motions appear to be crucial to impart the ferroxidase centers in BfrB with the flexibility necessary to capture and oxidize Fe 2+ , and subsequently allow passage of Fe 3+ into the central cavity. 8,47,48 It is therefore possible that seemingly subtle amino acid sequence differences between E. coli Bfr and P. aeruginosa BfrB impart the 24-mer proteins with different dynamic properties, which provide the corresponding ferroxidase centers with their distinct chemical properties. These unique properties in turn may be important determinants of the physiological role of the corresponding bacterioferritins.…”

Ferritins: furnishing proteins with iron

“…However, to date a full consideration of these issues has not been reported, doubtless a reflection of the difficulty in actually carrying out these kinds of studies. What has been established thus far is that the electrostatic profiles of channels through the protein shells of different ferritins differ considerably [ 4 , 16 , 97 , 98 ], and the dynamical properties of the channels of PaBFR [ 99 ] and the dynamical and electrostatic properties of frog ferritin [ 8 , 65 , 100 ] influence the manner in which these proteins accumulate iron. Similar studies with other ferritins are required to probe these aspects further.…”

Bacterioferritin: Structure, Dynamics, and Protein–Protein Interactions at Play in Iron Storage and Mobilization

“…24 Figure 9 shows a transverse view of the interior cavity in Fe-soaked C89S/K96C BfrB, with the 4-fold pores highlighted in purple, the 3-fold pores in blue, the B-pores in green, the ferroxidase pores in yellow, the iron ions in orange, and the phosphate ions in yellow and red. The view illustrates how iron can access the interior cavity via FCs and B-pores.…”