Investigation of the mechanism of iron release from the C-lobe of human serum transferrin: Mutational analysis of the role of a pH sensitive triad

The transferrins (TFs) are a family of proteins that are widely distributed in vertebrates, where they serve a major role in iron binding and transport. Most TFs are composed of two homologous lobes, the N- and C-lobes, each able to bind a single iron atom. Human serum transferrin (hTF) binds iron in the blood and delivers it to actively dividing cells; through the process of receptor-mediated endocytosis, diferric hTF in the serum (pH similar to7.4) binds to specific TF receptors on the cell surface and is internalized, whereupon a pH drop in the endosome (pH similar to5.6) facilitates iron release. Many factors affect the rate of iron release, including pH, chelator, temperature, salt, and lobe-lobe interactions. We, and others, have actively studied the mechanism of iron release from the recombinant N-lobe of hTF; in contrast, the exact details of iron release from the C-lobe have remained less well characterized but appear to differ from those found for the N-lobe. Recently, to simplify the purification protocol, we have expressed and purified full-length recombinant hTF containing an N-terminal hexahistidine tag [Mason et al. (2002) Biochemistry 41, 9448-9454]. In the present work, we have expressed a full-length recombinant hTF containing a K206E mutation such that the N-lobe does not readily release iron. The resulting full-length hTF allows us to focus on the C-lobe and to study the effects of mutations introduced into the C-lobe. The success of this strategy is documented and in vitro mutagenesis is used to identify three residues in the C-lobe that are critical for iron-release. Although the importance of this triad is unequivocally demonstrated, further studies are needed to completely elucidate the mechanism of iron release from the C-lobe of hTF. In addition, the striking difference in the effect of increasing salt concentrations on iron release from the two lobes of hTF is further documented in the present work.