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 C 14 D group in SRII K . Here, we identify the counterpart of the C 14 D group as Thr204, because the C 14 -D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C 14 -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 C 14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C 14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.Microbial rhodopsins convert light into chemiosmotic energy or cellular signals (1, 2). The primary reaction is all-trans to 13-cis photoisomerization of the retinal chromophore, and light energy is stored through altered chromophore-protein interaction (2, 3). Such energy is subsequently consumed by changing the protein structures, and ion pumping or transducer activation is achieved in light-energy or signal conversions, respectively. It is well-known that retinal isomerization in the protein is a selective and efficient reaction along the

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199(ppR)-Asn74(pHtrII) and Thr189(ppR)-Glu43 (pHtrII), Ser62(pHtrII). It was reported that a pHR mutant (P240T/F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L(1) and L(2) intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250(pHR) and Asn74(pHtrII) were detected for the L(1) and L(2) intermediates, supporting that Tyr250(pHR) forms a hydrogen bond with Asn74(pHtrII) as similarly to Tyr199(ppR). The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156(pHR) in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.

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