Zhang Lianyi, Koay Melissa, Maher Megan J, Xiao Zhiguang, Wedd Anthony G
School of Chemistry and Bio21 Institute, University of Melbourne, Parkville, VIC 3010, Australia.
J Am Chem Soc. 2006 May 3;128(17):5834-50. doi: 10.1021/ja058528x.
CopC is a small soluble protein expressed in the periplasm of Pseudomonas syringae pathovar tomato as part of its copper resistance response (cop operon). Equilibrium competition reactions confirmed two separated binding sites with high affinities for Cu(I) (10(-7) > or = K(D) > or = 10(-13) M) and Cu(II) (K(D) = 10(-13(1)) M), respectively. While Cu(I)-CopC was converted cleanly by O2 to Cu(II)-CopC, the fully loaded form Cu(I)Cu(II)-CopC was stable in air. Variant forms H1F and H91F exhibited a lower affinity for Cu(II) than does the wild-type protein while variant E27G exhibited a higher affinity. Cation exchange chromatography detected each of the four different types of intermolecular copper transfer reactions possible between wild type and variant forms: Cu(I) site to Cu(II) site; Cu(II) site to Cu(I) site; Cu(I) site to Cu(I) site; Cu(II) site to Cu(II) site. The availability of an unoccupied site of higher affinity induced intermolecular transfer of either Cu(I) or Cu(II) in the presence of O2 while buffering concentrations of cupric ion at sub-picomolar levels. Crystal structures of two crystal forms of wild-type Cu(I)Cu(II)-CopC and of the apo-H91F variant demonstrate that the core structures of the molecules in the three crystal forms are conserved. However, the conformations of the amino terminus (a Cu(II) ligand) and the two copper-binding loops (at each end of the molecule) differ significantly, providing the structural lability needed to allow transfer of copper between partners, with or without change of oxidation state. CopC has the potential to interact directly with each of the four cop proteins coexpressed to the periplasm.
CopC是一种小的可溶性蛋白，在丁香假单胞菌番茄致病变种的周质中表达，是其铜抗性反应（cop操纵子）的一部分。平衡竞争反应证实了存在两个对Cu(I)（10⁻⁷≥K(D)≥10⁻¹³ M）和Cu(II)（K(D)=10⁻¹³(1) M）具有高亲和力的分离结合位点。虽然Cu(I)-CopC能被O₂干净地转化为Cu(II)-CopC，但完全负载形式的Cu(I)Cu(II)-CopC在空气中是稳定的。变体形式H1F和H91F对Cu(II)的亲和力低于野生型蛋白，而变体E27G表现出更高的亲和力。阳离子交换色谱检测到野生型和变体形式之间可能发生的四种不同类型的分子间铜转移反应：从Cu(I)位点到Cu(II)位点；从Cu(II)位点到Cu(I)位点；从Cu(I)位点到Cu(I)位点；从Cu(II)位点到Cu(II)位点。在存在O₂的情况下，高亲和力的未占据位点的可用性会诱导Cu(I)或Cu(II)的分子间转移，同时将铜离子浓度缓冲在亚皮摩尔水平。野生型Cu(I)Cu(II)-CopC的两种晶体形式以及脱辅基H91F变体的晶体结构表明，三种晶体形式中分子的核心结构是保守的。然而，氨基末端（一种Cu(II)配体）和两个铜结合环（在分子的两端）的构象有显著差异，提供了在有或没有氧化态变化的情况下允许铜在伙伴之间转移所需的结构灵活性。CopC有可能与共表达于周质中的四种cop蛋白直接相互作用。
