Rammelsberg R, Huhn G, Lübben M, Gerwert K
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Biochemistry. 1998 Apr 7;37(14):5001-9. doi: 10.1021/bi971701k.
In its proton-pumping photocycle, bacteriorhodopsin releases a proton to the extracellular surface at pH 7 in the transition from intermediate L to intermediate M. The proton-release group, named XH, was assigned in low-temperature FT-IR studies to a single residue, E204 [Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R. , and Lanyi, J. K. (1995) J. Biol. Chem. 270, 27122-27126]. The time-resolved room-temperature step-scan FT-IR photocycle studies on wild-type and E204Q-, and E204D-mutated bacteriorhodopsin, which we present here, show in contrast that the FT-IR data give no evidence for deprotonation of E204 in the L-to-M transition. Therefore, it is unlikely that E204 represents XH. On the other hand, IR continuum absorbance changes indicate intramolecular proton transfer via an H-bonded network to the surface of the protein. It appears that this H-bonded network is spanned between the Schiff base and the protein surface. The network consists at least partly of internally bound water molecules and is stabilized by E204 and R82. Other not yet identified groups may also contribute. At pH 5, the intramolecular proton transfer to the surface of the protein seems not to be disturbed. The proton seems to be buffered at the surface and later in the photocycle released into the bulk during BR recovery. Intramolecular proton transfer via a complex H-bonded network is proposed to be a general feature of proton transfer in proteins.
在其质子泵浦光循环中，细菌视紫红质在从中间体L转变为中间体M的过程中，于pH 7时向细胞外表面释放一个质子。在低温傅里叶变换红外光谱（FT-IR）研究中，被命名为XH的质子释放基团被确定为单个残基E204 [布朗，L. S.，佐佐木，J.，神鸟，H.，前田，A.，尼德曼，R.，以及兰伊，J. K.（1995年）《生物化学杂志》270，27122 - 27126]。然而，我们在此展示的对野生型、E204Q和E204D突变型细菌视紫红质进行的时间分辨室温步进扫描FT-IR光循环研究表明，FT-IR数据没有提供E204在L到M转变过程中去质子化的证据。因此，E204不太可能代表XH。另一方面，红外连续吸收变化表明分子内质子通过氢键网络转移到蛋白质表面。看来这个氢键网络跨越在席夫碱和蛋白质表面之间。该网络至少部分由内部结合的水分子组成，并由E204和R82稳定。其他尚未确定的基团可能也有贡献。在pH 5时，分子内向蛋白质表面的质子转移似乎未受干扰。质子似乎在表面被缓冲，随后在光循环后期细菌视紫红质恢复过程中释放到主体中。通过复杂氢键网络进行的分子内质子转移被认为是蛋白质中质子转移的一个普遍特征。
