The Role Of Arginine 83 In Light-driven Proton Pump Archearhodopsin-4

Archaerorhodopsin-4(aR4)is a photoreceptor containing carotenoid chromophore in Halobacterium species XZ515(H.sp.XZ515).It has a similar trimeric structure and a light-driven proton pump function as bacteriorhodopsin(b R).After being excited by light,the retinal is isomerized from all-trans to 13-cis and enters the photocycle.After going through a series of intermediate states with specific wavelengths such as K,L,M,N,and O,the protein returns to the ground state,accompanied by the directional transfer of protons from the intracellular to the extracellular.Although both aR4 and b R are outward proton pumps,aR4 exhibits a reversed temporal order of proton release and uptake compared with b R.Arginine-82,located in the extracellular half channel of b R,plays an important role in proton transfer,and 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 b R.So far,nothing is clear about this orientation difference with the temporal order of proton release.This paper uses genetic engineering,UV-Vis absorption spectroscopy,flash-light induced photolysis spectroscopy and p H titration measurements to compare the function difference of R83 in aR4 and R82 in b R to explore the function of R83 in aR4 and whether the bacterioruberin affects the functional role of R83.The results show that R83 also act as a "bridge" to link the proton acceptor and PRC in aR4,but the orientation of R83 is more intracellular than that of b R,and the distance between R83 and PRC is farther.Therefore,the hydrogen bond interaction of R83 with PRC is weaker than R82 with PRC in b R,which might be one of the reasons for the different temporal order of proton release in aR4.In addition,bacterioruberin is a hydrophobic long-chain sterol that is embedded between aR4 trimers.It can stabilize the trimeric structure,removing bacterioruberin will cause protein structure to be loosely assembled,reduce the uniformity of the membrane layer,weaken the effect of R83 mutation on protein structure and function,and increase the p H dependence of the R83 function.