Thermodynamic Analysis of Ferrous Ion Binding toEscherichia coliFerritin EcFtnA

Abstract

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 Fe 2+ 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 Fe 2+ 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 Fe 2+ -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 Fe 2+ , both A and B sites in EcFtnA bind Fe 2+ , implying a role for the C site in influencing the binding of Fe 2+ 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.

Calorimetric and Spectroscopic Studies of Hoechst 33258: Self-association and Binding to Non-cognate DNA

“…(2) without the need for tedious algebraic combination of the equations describing individual equilibria. 48 The calculation of concentrations outlined above is repeated for the ITC cell solution after every injection i and for the solution in the injection syringe (interactions occurring in the injection syringe are treated in exactly the same way as those in the sample cell, complex titrant solutions can therefore be dealt with routinely). These concentrations are then used to calculate "concentrations of interactions" [int] X (see Supplementary Data).…”

Structural and Thermodynamic Characterization of Metal Ion Binding in Streptococcus suis Dpr

“…In most cases, binding was mainly driven by entropy, as seen with ferritins, L. innocua Dps, and Escherichia coli frataxin. [22][23][24][25][26] The favorable entropy is probably contributed by desolvation, as water molecules are released from the hydration layer around the metal ions and from the FOC as binding occurs.…”

The Crystal Structure of Ferritin from Helicobacter pylori Reveals Unusual Conformational Changes for Iron Uptake

“…This region on the inner surface has been considered as a possible iron nucleation center in Ecf and as an iron nucleation site to complex a metal ion in a roughly tetrahedral geometry in HuHf. 9,18 It is thus highly likely that ferritins have a common inner nucleation site mediated by the conserved glutamate residues, although Glu130 is only conserved in prokaryotes (Fig. 1a).…”