Effect of Carotenoid Chromophore on the Function of the Key Residue F146 in the Light-driven Proton Pump Archaerhodopsin-4

Archaerhodopsin-4（aR4）, a bacteriorhodopsin（bR） like light-driven proton pump, possesses the similar trimeric structure. aR4 contains not only an opsin molecule and a covalently bound retinal chromophore, but also a carotenoid chromophore bacterioruberin that is packed in the crevices between the adjacent trimer subunits of the protein trimer. In addition, aR4 displays an opposite temporal order of proton release and uptake to that of bR. Phenylalanine-146（F146） and methionine-145（M145） are the key residues that locate within the same position in the retinal binding pocket of aR4 and bR, respectively, and the only different residue of the two binding pockets. It was reported that M145 F mutation can affect the cis-trans thermal equilibrium of retinal in the dark-adapted bR, and has an important effect on its photocycle. However, nothing is clear about the function of F146 in the proton pump aR4, especially any modulation of the carotenoid chromophore bacterioruberin to the function of the key residue in the retinal binding pocket. To address this research gap, site-specific mutagenesis, combined with in situ UV-Vis absorption spectroscopy, solid-state NMR, flash light-induced transient absorption change measurements, and pH titration were used to study the function commonality and difference of F146 M and M145 F mutation in aR4 and bR. Our results showed that both F146 and M145 played a role in maintaining the cis-trans thermal equilibrium of retinal in the dark-adapted proteins. Both F146 M and M145 F mutations could cause changes to the retinal binding pocket and the hydrogen bond network with the proton translocation channel, thus participating in the regulation of proton transfer. In addition, it was identified that bacterioruberin not only can stabilize the trimeric structure, but also can modulate the function of F146, a key residue of the retinal binding pocket, removal of bacterioruberin will weaken the effect of F146 in aR4.