FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 isthe Schiff base proton acceptor

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halob acterium salinarum, has several homologous residues which have been found t o be important for the proper functioning of bacteriorhodopsin (BR), a ligh t-driven proton pump. These include Asp73, which in the case of bacteriorho dopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, bo th reconstituted in Halobacterium salinarum lipids, using FTIR difference s pectroscopy under conditions that favor accumulation of the O-like, photocy cle intermediate, SII540. At both room temperature and -20 degrees C, the d ifference spectrum of SRII is similar to the BR-->O-640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint r egion. This indicates that SII540 has an all-trans chromophore similar to t he O-640 intermediate in BR. A positive band at 1761 cm(-1) downshifts 40 c m(-1) in the mutant D73E, confirming that Asp73 undergoes a protonation rea ction and functions in analogy to Asp85 in BR as a Schiff base proton accep tor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or G lu residues. Intense bands in the amide I region indicate that a protein co nformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. Howeve r, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these ban ds are partially accessible for hydrogen/deuterium exchange. Implications o f these findings for the mechanism of SRII signal transduction are discusse d.