D97N突变对光受体蛋白古紫质4质子泵和能量转换效率的影响

Effect of D97N Mutation on Proton Transport and Energy Conversion in the Photoreceptor Archaerhodopsin 4

古紫质4(archaerhodopsin 4,aR4)是新近发现的古生菌Halobacterium species xz515红膜上唯一的光敏视黄醛蛋白,具有和细菌视紫红质(Bacteriorhodopsin,bR)相似的质子泵功能,但在中性p H条件下,其质子释放和摄取顺序与bR相反。针对质子供体天冬氨酸97(Aspartic acid 97,D97)对aR4光循环;特别是对质子释放摄取顺序和菌株ATP生成率的影响,采用基因定点突变技术,构建了aR4的单突变体D97N,以及相对应的bR单突变体D96N。采用紫外-可见吸收光谱和闪光动力学光谱技术初步研究突变对视黄醛键合区、光反应中间态M态和O态、质子泵功能以及菌株ATP生成率的影响。结果表明,D97N突变对视黄醛紫外-可见光吸收波长没有太大影响,但造成M态衰减时间的显著延长、质子泵功能的消失及菌株ATP的生成率大幅降低。与bR中的D96作用相比,D97对aR4质子功能的影响有所不同,这可能与D97所处的一个更为疏水性的胞外侧环境有关。

Archaerhodopsin 4（ aR4） is a newly discovered photosensitive retinal protein from the claret membrane of Halobacterium species xz515. It functions as a proton pump similar to bacteriorhodopsin（ bR） but with an opposite temporal order of proton uptake and release at neutral pH. Aspartic acid 97（ D97） is one of the key residues as the proton donor during the photocycle in aR4. Its function on the photocycle,the proton pump function and the energy conversion is very important for a deep understanding of the relationships between aR4 structure and function,especially with proton release and uptake order. Single mutation of D96 N of bR and D97 N of aR4 have been successfully constructed by combining site-directed mutagenesis with heterologous expression in Halobacterium species L33. The influences of mutations on the retinal binding pocket,transient kinetic change of the M state and O state,and the ATP conversion efficiency have been studied by UV-VIS and flashlight induced kinetic absorption change spectroscopy. The results show that D97 N mutation in aR4 does not affect the UV absorption of the retinal chromophore,but results in a significant prolongation of the M state,a greatly weakened proton pump,and a decreased ATP formation efficiency. This indicates a different function of D97 in aR4 than the same type residue D96 in bR due to a more hydrophobic environment along the proton translocation channel from D97 to the retinal chromophore.