Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA.
Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.
Bou-Abdallah
Fadi
F
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, USA. fadib@cisunix.unh.edu
Woodhall
Mark R
MR
Velázquez-Campoy
Adrián
A
Andrews
Simon C
SC
Chasteen
N Dennis
ND
D002151:Calorimetry; D005293:Ferritins / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D013816:Thermodynamics
D005296:Ferrous Compounds; D005293:Ferritins