Inequivalence of the two 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 present 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 liganding tyrosines were mutated individually to phenylalanine, the resulting 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 aspartate, did not completely abolish iron binding, the present findings suggest that the tyrosine ligand at position 188 is essential for binding of iron to occur. Replacement of Tyr 188 with phenylalanine created a favorable chemical environment for copper coordination but a fatal situation for iron binding. The positions of the two liganding tyrosines in the metal-binding cleft suggest a reason for the inequivalence.
He
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QY
Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405, USA. qhe@zoo.uvm.edu
Mason
A B
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Woodworth
R C
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Tam
B M
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MacGillivray
R T
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Grady
J K
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Chasteen
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ND
D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000737:chemistry; D008024:Ligands; D016296:Mutagenesis; D009571:Nitrilotriacetic Acid; D011994:Recombinant Proteins / Q000737:chemistry; D017186:Titrimetry; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D008024:Ligands; D011994:Recombinant Proteins; D014168:Transferrin; D001224:Aspartic Acid; D014443:Tyrosine; D003300:Copper; D007501:Iron; D009571:Nitrilotriacetic Acid