Deprotonation of retinal Schiff base and structural dynamics in the early photoreaction of primate blue cone visual pigment.

Animal rhodopsin is a photoreceptive protein crucial for vision, with activation triggered by the cis-trans isomerization of a retinal chromophore upon light absorption. This activation involves a series of thermal intermediates, ultimately leading to G protein-mediated signal transduction. The retinal chromophore is covalently bound to the protein through a protonated Schiff base, and its deprotonation during the formation of the active intermediate is believed to induce structural changes in α-helices that facilitate G-protein interactions. Using low-temperature UV-visible absorption and Fourier transform infrared spectroscopy, we investigated the early photoreaction of the primate blue cone visual pigment (MB). Our results demonstrate that Schiff base deprotonation in the early photoreaction is coupled with local perturbations in α-helices, promoting the formation of the Lumi intermediate. Using site-directed mutagenesis, we identified the proton acceptor involved in Schiff base deprotonation and mapped the regions of α-helical structural changes during the formation of the Lumi intermediate. We discovered that the proton released from the Schiff base is transferred to the counterion Glu113. Systematic mutagenesis revealed that structural perturbations in transmembrane helix 7 bring Glu113 and the lysine residue forming the Schiff base into proximity, facilitating efficient proton transfer during the early photoreaction. Additionally, the Lumi intermediate formed at low temperatures was found to revert to the original state through thermally driven reverse proton transfer, coupled with retinal reisomerization. From an evolutionary perspective, MB is part of a group of UV-sensitive cone visual pigments characterized by a deprotonated retinal Schiff base in the ground state. The observed propensity for MB to undergo Schiff base deprotonation is consistent with this evolutionary trait.