title: The Schiff base counterion of bacteriorhodopsin is protonated in sensory rhodopsin I: spectroscopic and functional characterization of the mutated proteins D76N and D76A.
abstract: Both sensory rhodopsin I (SR-I), a phototaxis receptor, and bacteriorhodopsin (BR), a light-driven proton pump, share residues which have been identified as critical for BR functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions both as the Schiff base counterion and proton acceptor. We found that substituting an Asn for Asp76 (D76N) in SR-I has no effect on its visible absorption unlike the analogous mutation (D85N) in BR which shifts the absorption to longer wavelengths. The mutated proteins D76N and D76A are also fully functional as phototaxis receptors in contrast to BR, where the analogous substitutions block proton transport. D76N was also found to exhibit a spectrally normal SR587-->S373 transition. However, FTIR difference spectroscopy reveals that two bands in the SR587-->S373 difference spectrum at 1766/1749 cm-1 (negative/positive), assigned to the C=O stretch mode of a carboxylic acid, disappear in D76N, although no changes are observed in the carboxylate region. In addition, the kinetics and yield of this photoreaction are altered. On this basis, it is concluded that, unlike Asp85 in bacteriorhodopsin, Asp76 is protonated in SR-I and undergoes an increase in its hydrogen bonding during the SR587-->S373 transition. This model accounts for the difference in color of SR-I and BR and the finding that Asn can substitute for Asp76 without greatly altering the SR-I phenotype. Interestingly, parallels exist between this residue and Asp83 in the visual receptor rhodopsin which has recently been found to exist in a protonated form and to undergo an almost identical change in hydrogen bonding during rhodopsin activation.
authors.affiliation: Department of Physics, Boston University, Massachusetts 02215.
authors.forename: P
authors.initials: P
authors.lastname: Rath
authors.forename: K D
authors.initials: KD
authors.lastname: Olson
authors.forename: J L
authors.initials: JL
authors.lastname: Spudich
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002465:Cell Movement / Q000528:radiation effects; D017931:DNA Primers; D004926:Escherichia coli; D006217:Halobacterium; D025602:Halorhodopsins; D008969:Molecular Sequence Data; D009154:Mutation; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry*; D025603:Sensory Rhodopsins; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis
chemical_list: D017931:DNA Primers; D025602:Halorhodopsins; D011522:Protons; C061497:SRI protein, Halobacterium; D012545:Schiff Bases; D025603:Sensory Rhodopsins; D001436:Bacteriorhodopsins