title: Asp76 is the Schiff base counterion and proton acceptor in the proton-translocating form of sensory rhodopsin I.
abstract: Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light-driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton-transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin.
authors.affiliation: Department of Physics, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: P
authors.initials: P
authors.lastname: Rath
authors.forename: E
authors.initials: E
authors.lastname: Spudich
authors.forename: D D
authors.initials: DD
authors.lastname: Neal
authors.forename: J L
authors.initials: JL
authors.lastname: Spudich
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
mesh_terms: D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D003624:Darkness; D025602:Halorhodopsins; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012545:Schiff Bases; D011984:Sensory Receptor Cells / Q000737:chemistry / Q000378:metabolism; D025603:Sensory Rhodopsins; D016324:Sequence Tagged Sites; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D025602:Halorhodopsins; D011994:Recombinant Proteins; C061497:SRI protein, Halobacterium; D012545:Schiff Bases; D025603:Sensory Rhodopsins; D001224:Aspartic Acid; D006639:Histidine; D001436:Bacteriorhodopsins