Direct thermodynamic and kinetic measurements of Fe2 + and Zn2 + binding to human serum transferrin

Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of FeII to FeIII and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe2 + and Zn2 + ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2 nM for Fe2 + and 6.7 nM for Zn2 + and maximum binding stoichiometries of 1 Zn2 + (or 1 Fe2 +) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH0 ~ − 12 kJ/mol and ΔS0 ~ 106 J/mol·K for Fe2 + and ΔH0 ~ − 18 kJ/mol and ΔS0 ~ 97 J/mol·K for Zn2 +). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe3 +–hTf complex might be reduced to the more labile Fe2 + ion before iron is released to the cytosol.

Graphical abstractThermodynamics of Fe2 + and Zn2 + association with human serum transferrinFigure optionsDownload full-size imageDownload as PowerPoint slide