He Q Y, Mason A B, Woodworth R C, Tam B M, MacGillivray R T, Grady J K, Chasteen N D
Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405, USA.
Biochemistry. 1997 Dec 2;36(48):14853-60. doi: 10.1021/bi9719556.
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.
人血清转铁蛋白N叶（hTF/2N）有四个铁结合配体，包括一个组氨酸、一个天冬氨酸和两个酪氨酸。本报告通过定点诱变、金属释放动力学以及吸收光谱和电子顺磁共振（EPR）光谱，阐明了两个酪氨酸配体（Tyr 95和Tyr 188）在hTF/2N金属结合特性上的不等效性。当配位酪氨酸分别突变为苯丙氨酸时，产生的突变体Y95F对铁的结合亲和力较弱，对铜无亲和力，而突变体Y188F完全丧失了结合铁的能力，但与铜形成了稳定的复合物。由于其他研究表明，另外两个配体组氨酸和天冬氨酸的突变并没有完全消除铁结合，因此本研究结果表明，188位的酪氨酸配体对于铁的结合至关重要。用苯丙氨酸取代Tyr 188为铜配位创造了有利的化学环境，但对铁结合却是致命的情况。两个配位酪氨酸在金属结合裂隙中的位置说明了这种不等效性的原因。
