Karlsson B G, Tsai L C, Nar H, Sanders-Loehr J, Bonander N, Langer V, Sjölin L
Department of Biochemistry and Biophysics, Goteborg University, Sweden.
Biochemistry. 1997 Apr 8;36(14):4089-95. doi: 10.1021/bi962416o.
The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.
Met121Glu 型天青蛋白突变体已被结晶，其结构在 2.3 Å 的分辨率下得以确定。在晶体结构中，Met121Glu 的一个羧基氧与金属配位，距离为 2.2 Å。利用单晶共振拉曼光谱表明铜位点的谷氨酸残基处于质子化状态。对该残基进行滴定会产生许多不寻常的、pH 依赖性特性：随着 pH 从 4 增加到 8，S(Cys)-Cu 配体到金属的电荷转移带发生蓝移且其强度比反转，电子顺磁共振 (EPR) 信号从 1 型铜转变为一种新形式的蛋白质结合铜，氧化还原电位从 370 mV 变为 180 mV。此 pH 区间内的光谱变化与双态模型一致。根据光学和 EPR 光谱的 pH 依赖性，确定氧化态蛋白质中谷氨酸的 pKa = 5.0。
