Steric constraint in the primary photoproduct of sensory rhodopsin II is a prerequisite for light-signal transfer to HtrII

Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C ₁₄-D stretching vibration of the retinal chromophore at 2244 cm ^-1 upon formation of the K state and interpreted that a steric constraint occurs at the C₁₄D group in SRII_K. Here, we identify the counterpart of the C₁₄D group as Thr204, because the C₁₄-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C₁₄-HOOP (hydrogen out-of-plane) vibration at 864 cm^-1. Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm^-1 bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C₁₄ atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C₁₄ group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.