Chon Y S, Sasaki J, Kandori H, Brown L S, Lanyi J K, Needleman R, Maeda A
Department of Biophysics, Graduate School of Science, Kyoto University, Japan.
Biochemistry. 1996 Nov 12;35(45):14244-50. doi: 10.1021/bi9606197.
The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide binds near the Schiff base. In D85T this occurs near neutral pH, but in D85N only at pH < 4. The structures of the L and the unphotolyzed states of these proteins were examined with Fourier transform infrared spectroscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the wild-type protein. Shift of the ethylenic band toward lower frequency upon replacing chloride by bromide confirmed the contribution of the negative charge of the anions to the Schiff base counterion. These anions restored the change of water, which is bound near the protonated Schiff base but is absent in the blue form of the D85N mutant, though with stronger H-bonding than in the wild type. The C = N stretching vibration of the Schiff base in H2O and 2H2O was detected by Fourier transform Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to the mutants than with Asp85 as the counterion in the wild type. Thus, although the geometry of the environment is different, there is at least one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.
细菌视紫红质的D85T和D85N突变体的发色团呈蓝色，但当氯离子或溴离子在席夫碱附近结合时会变成像野生型一样的紫色。在D85T中，这种情况发生在接近中性pH时，但在D85N中仅在pH < 4时发生。用傅里叶变换红外光谱研究了这些蛋白质的L态和未光解态的结构。紫色形式的差谱，而不是在没有这些阴离子时的蓝色形式，类似于野生型蛋白质的光谱。用溴离子取代氯离子时烯键带向低频的移动证实了阴离子的负电荷对席夫碱抗衡离子的贡献。这些阴离子恢复了水的变化，水在质子化席夫碱附近结合，但在D85N突变体的蓝色形式中不存在，尽管其氢键比野生型更强。通过傅里叶变换拉曼光谱检测了H2O和2H2O中席夫碱的C = N伸缩振动。当氯离子或溴离子与突变体结合时，未光解态的席夫碱的氢键强度比野生型中以Asp85作为抗衡离子时弱。因此，尽管环境的几何形状不同，但在这些突变体中至少有一个水分子以类似于野生型中与Asp85结合的水的方式与结合的卤化物配位。
