精氨酸82和精氨酸83在光驱质子泵细菌视紫红质和古紫质-4中的功能对比研究

Comparative Study of the Function of Arginine 82 and Arginine 83 in the Light-driven Proton Pumps Bacteriorhodopsin and Archaerhodopsin-4

古紫质-4(Archaerhodopsin-4,aR4)是一种存在于嗜盐菌Halobacterium species XZ515细胞膜上含有类胡萝卜素发色团菌红素(Bacterioruberin)的光敏受体蛋白,与细菌视紫红质(Bacteriorhodopsin,bR)具有相似的三聚结构和光驱质子泵功能。尽管二者均为外向质子泵,但aR4表现为与bR不同的先摄取后释放的质子传输时序。位于bR胞外半通道的精氨酸82(Arginine 82,R82)充当着视黄醛键合区五边形氢键网络与质子释放簇(proton release cluster,PRC)之间的桥梁,不仅向胞外传递D85的质子化信息,同时调控PRC质子的释放和再质子化。R83作为aR4中的同源等位残基,其侧链取向与bR中的R82有所不同,而这一差异是否与其质子释放时序的不同有关目前尚未见报道。为此,本研究采用定点突变技术、原位紫外-可见光吸收光谱、闪光动力学光谱、酸碱滴定等技术手段,对比分析R83和R82在aR4和bR中的差异性作用。研究表明,R83和R82在aR4和bR中均起到质子受体与质子释放簇之间的“桥梁”作用,但是R83与PRC间的氢键相互作用要弱于R82与PRC间的氢键相互作用,这可能是导致aR4不同质子释放时序的原因之一。此外,菌红素的缺失可导致R83功能对体系pH依赖性增加。

Archaerorhodopsin-4(aR4)is a photoreceptor containing carotenoid chromophore from Halobacterium species XZ515.It has a similar trimeric structure and a light-driven proton pump function as bacteriorhodopsin(bR).Although both aR4 and bR are outward proton pumps,aR4 exhibits a reversed temporal order of proton release and uptake compared with bR.Arginine 82,located in the extracellular half channel of bR,bridges the connection between the retinal binding pocket and proton release cluster(PRC).It propagates the protonation status of D85 to the extracellular side and regulates proton release and reprotonation of PRC.R83 is the corresponding residue in aR4 but has a different orientation for its sidechain compared with R82 in bR.So far,nothing is clear about this orientation difference with the temporal order of proton release.This paper uses site-specific mutagenesis,combined with in situ UV-Vis absorption spectroscopy,flash light-induced transient absorption change measurements,and acid-base titration to explore the function difference of R83 in aR4 and R82 in bR.The results show that both R83 and R82 act as a"bridge"to link the proton acceptor and PRC in two proteins,but the hydrogen bond interaction of R83 with PRC is weaker than R82 with PRC in bR,which might be one of the reasons for the different temporal order of proton release in aR4.In addition,removing bacterioruberin in aR4 will increase the pH dependence of the R83 function.