Shongwe M S, Smith C A, Ainscough E W, Baker H M, Brodie A M, Baker E N
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Biochemistry. 1992 May 12;31(18):4451-8. doi: 10.1021/bi00133a010.
The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.
通过物理化学和晶体学技术研究了乳铁蛋白（Lf）与Fe3+或Cu2+作为相关金属离子时的阴离子结合特性。这些研究突出了两个位点之间以及不同金属结合时阴离子结合行为的差异。制备了碳酸盐、草酸盐以及碳酸盐 - 草酸盐混合配合物，并记录了它们的特征电子光谱和电子顺磁共振光谱。根据草酸盐浓度，草酸盐可以从Cu2(CO3)2Lf的一个或两个阴离子位点取代碳酸盐，但Fe2(CO3)2Lf不会发生这种取代。在无碳酸盐条件下向脱铁乳铁蛋白中添加草酸盐和适当的金属离子会形成Fe3+和Cu2+的二草酸盐配合物，除非蛋白质中残留痕量的EDTA，此时可能会形成混合配合物M2(CO3)(C2O4)Lf。分别在2.2 Å、2.1 Å和2.2 Å分辨率下精修的Fe2(CO3)2Lf、Cu2-(CO3)2Lf和Cu2(CO3)(C2O4)Lf晶体结构中的阴离子位点已进行了比较。在每种情况下，阴离子都通过氢键与螺旋5的N端、一个相关的精氨酸侧链以及附近的苏氨酸侧链相连。碳酸根离子以双齿方式与金属结合，但在双铜乳铁蛋白的N叶位点中为单齿结合；这种差异源于金属离子的轻微移动。混合配合物表明草酸根离子优先以1,2 - 双齿模式结合在C叶位点，但会使附近的几个侧链发生位移。这些观察结果得出了转铁蛋白协同阴离子结合的通用模型。
