Crystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein

The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe3+-binding protein (hFbp) has been determined to 1.75 Angstrom resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K-d of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe3+, while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe3+ ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe3+-binding proteins for Fe3+ (nFbp K'(eff) = 2.4 x 10(18) M-1). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe3+-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.