Groeneveld C M, Ouwerling M C, Erkelens C, Canters G W
Gorlaeus Laboratories, Leiden University, The Netherlands.
J Mol Biol. 1988 Mar 5;200(1):189-99. doi: 10.1016/0022-2836(88)90343-9.
The proton nuclear magnetic resonance spectrum of azurin from Alcaligenes denitrificans at pH 6.0 and 309 K is reported. Proton signals from all methionine and histidine residues (among them the copper ligands) have been assigned. The data have been used to study the pH behaviour of His35 and to establish the electron self-exchange rate of the protein. His35 appears to be protonated at pH less than 4.5, possibly after rupture of a salt bridge. No effects of this protonation on the tertiary structure around the copper site are observed, however, contrary to the case of Pseudomonas aeruginosa azurin. The electron self-exchange rate amounts to 4 x 10(5) M-1 S-1 at pH 6.7 and 297 K. The data support the conclusion that the electron self-exchange takes place by way of the hydrophobic surface patch around His117, and that His35 is not involved in this reaction. Oxidation of azurin increases the acidity of the freely titrating His32 and His83 by 0.07 and 0.25 pKa units, respectively. The data can be used to test the theory of electrostatic interactions in proteins. The optical extinction coefficient at 625 nm was experimentally determined and amounts to 4.8(+/- 0.1) x 10(3) M-1 cm-1.
报道了反硝化产碱菌中铜蓝蛋白在pH 6.0和309 K时的质子核磁共振谱。已对所有甲硫氨酸和组氨酸残基（包括其中的铜配体）的质子信号进行了归属。这些数据已用于研究His35的pH行为并确定该蛋白的电子自交换速率。His35在pH小于4.5时似乎被质子化，可能是在盐桥断裂之后。然而，与铜绿假单胞菌铜蓝蛋白的情况相反，未观察到这种质子化对铜位点周围三级结构的影响。在pH 6.7和297 K时，电子自交换速率为4×10⁵ M⁻¹ s⁻¹。这些数据支持以下结论：电子自交换通过His117周围的疏水表面区域发生，且His35不参与此反应。铜蓝蛋白的氧化分别使可自由滴定的His32和His83的酸度增加0.07和0.25个pKa单位。这些数据可用于检验蛋白质中静电相互作用的理论。通过实验测定了625 nm处的光消光系数，其值为4.8（±0.1）×10³ M⁻¹ cm⁻¹。
