Effect Of D97N Mutation On Proton Transport And Energy Conversion In The Photoreceptor Archaerhodopsin 4

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.A deep study of the influence of aspartic acid 97(D97),the proton donor of aR4,on its photocycle,proton pump and energy conversion is very important to reveal the opposite temporal order of proton uptake and release during aR4 photocycle at the molecular level.In this study,mutations of D96N of bR and D97N of aR4 have been successfully constructed by combining site-directed mutagenesis with heterologous expression in Halobacterium species L33.In situ measurements of solid-state nuclear magnetic resonance(ssNMR),combined with flashlight induced kinetic absorption change spectroscopy,UV-VIS spectroscopy and low-temperature transmission FTIR difference spectroscopy have been carried out to elucidate the influences of the mutations on the retinal binding pocket,photo-intermediates,proton pump and ATP conversion efficiency in the native membrane environment.Flashlight induced kinetic absorption change measurements show that D97N mutation of aR4 does not affect the retinal chromophore,but results in a significant prolongation of the M state and a greatly weakened proton pump.Whereas,D96N mutation of bR causes prolongation of the M state and loss of the O state.ssNMR experiments,together with UV-VIS,dynamic light scattering and ATP formation rate measurements show that both D97 of aR4 and D96 of bR have not much influence on the retinal binding pocket due to a long distance of 10 ? far away from each other.However,it has effects on the proton pump and protein packing,resulting in a decrease of the ATP formation efficiency by 50%.Low-temperature transmission FTIR results show that the process of protonation of D86 occurs at the L state and maintains until the N state,much early happened then the process of D85 in bR.All the results indicate a different function of D97,the proton donor of aR4 by comparing with the function of D96 in bR,suggesting different photocycle mechanisms between aR4 and bR,which corresponds directly to the opposite temporal order of proton uptake and release by the two proteins.