INEQUIVALENCE OF THE 2 TYROSINE LIGANDS IN THE N-LOBE OF HUMAN SERUM TRANSFERRIN

Human serum transferrin N-lobe (hTF/2N) has four iron-binding ligands, including one histidine, one aspartate, and two tyrosines. The presen t report elucidates the inequivalence of the two tyrosine ligands (Tyr 95 and Tyr 188) on the metal-binding properties of hTF/2N by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. When the ligand ing tyrosines were mutated individually to phenylalanine, the resultin g mutant Y95F showed a weak binding affinity for iron and no affinity for copper, whereas, mutant Y188F completely lost the ability to bind iron but formed a stable complex with copper. Since other studies have demonstrated that mutations of the other two ligands, histidine and a spartate, did not completely abolish iron binding, the present finding s suggest that the tyrosine ligand at position 188 is essential for bi nding of iron to occur. Replacement of Tyr 188 with phenylalanine crea ted a favorable chemical environment for copper coordination but a fat al situation for iron binding. The positions of the two liganding tyro sines in the metal-binding cleft suggest a reason for the inequivalenc e.