Structural changes of sensory rhodopsin I and its transducer protein are dependent on the protonated state of Asp76.

Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S 373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm (-1), and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm (-1) (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized.