NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, IL 60439, USA.
J Inorg Biochem. 2011 Dec;105(12):1638-44. doi: 10.1016/j.jinorgbio.2011.08.002. Epub 2011 Aug 12.
The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn(2+) at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn(2+). Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.
I型铜蛋白蓝血蛋白轴向配体的突变从蛋氨酸到赖氨酸导致蛋白质在光谱上不可见且氧化还原失活。M98K 蓝血蛋白作为竞争性抑制剂在天然蓝血蛋白与甲胺脱氢酶的反应中起作用，表明 M98K 突变并未影响其天然电子供体的亲和力。M98K 蓝血蛋白的晶体结构表明，其整体结构与天然蓝血蛋白非常相似，但 I 型结合位点被锌占据。使用在铜和锌吸收边缘附近收集的数据计算的异常差傅里叶图证实 I 型位点存在 Zn(2+)。Lys98 NZ 向 I 型位点的有序水分子提供氢键，增强了 Lys98 为 Zn(2+)提供配体的能力。尝试用铜重新生成 M98K 脱辅基蓝血蛋白导致蛋白质沉淀。M98K 突变产生如此具有选择性的锌结合蛋白令人惊讶，因为赖氨酸的锌配位很少见，这种配体集对于锌是独特的。
