Effect of genetic modification of tyrosine-185 on the proton pump and the blue-to-purple transition in bacteriorhodopsin.

The retinylidene chromophore mutant (Y185F) of bacteriorhodopsin, in which Tyr-185 is substituted by phenylalanine, is examined and compared with wild-type bacteriorhodopsin expressed in Escherichia coli; both were reinstituted similarly in vesicles. The Y185F mutant shows (at least) two distinct spectra at neutral pH. Upon light absorption, the blue species (which absorbs in the red) behaves as if "dead"--i.e., neither its tyrosine nor its protonated Schiff base undergoes deprotonation nor does its tryptophan fluorescence undergo quenching. This result is unlike either the purple species (which absorbs in the blue) or wild-type bacteriorhodopsin expressed in E. coli. As the pH increases, both the color changes and the protonated Schiff base deprotonation efficiency suggest a blue-to-purple transition of the Y185F mutant near pH 9. If this blue-to-purple transition of Y185F corresponds to the blue-to-purple transition of purple-membrane (native) bacteriorhodopsin (occurring at pH 2.6) and of wild-type bacteriorhodopsin expressed in E. coli (occurring at pH 5), the protein-conformation changes of this transition as well as the protonated Schiff base deprotonation may be controlled not by surface pH alone, but rather by the coupling between surface potential and the general protein internal structure around the active site. The results also suggest that Tyr-185 does not deprotonate during the photocycle in purple-membrane bacteriorhodopsin.

本研究对酪氨酸185被苯丙氨酸取代的细菌视紫红质（bacteriorhodopsin）视黄亚基生色团突变体（Y185F）进行了检测，并与在大肠杆菌（Escherichia coli）中表达的野生型细菌视紫红质进行比对；二者均以相似方式重组于囊泡中。Y185F突变体在中性pH条件下至少呈现两种不同的光谱特征。经光吸收作用后，吸收红光的蓝色物种表现出“失活”特征——其酪氨酸残基与质子化希夫碱（protonated Schiff base）均未发生去质子化，色氨酸荧光（tryptophan fluorescence）也未出现淬灭。这一现象与吸收蓝光的紫色物种，以及在大肠杆菌中表达的野生型细菌视紫红质均不相同。随着pH升高，从颜色变化与质子化希夫碱去质子化效率两方面来看，Y185F突变体在pH≈9附近发生了蓝色向紫色的转变。若将Y185F的该蓝-紫转变，与紫色膜（天然）细菌视紫红质（发生于pH 2.6）以及在大肠杆菌中表达的野生型细菌视紫红质（发生于pH 5）的蓝-紫转变相对应，则该转变过程中的蛋白质构象变化与质子化希夫碱去质子化过程，可能并非仅由表面pH调控，而是由表面电势与活性位点（active site）附近的蛋白质整体内部结构之间的耦合作用所调控。本研究结果同时表明，在紫色膜细菌视紫红质的光循环（photocycle）过程中，酪氨酸185并未发生去质子化。