THE SCHIFF-BASE COUNTERION OF BACTERIORHODOPSIN IS PROTONATED IN SENSORY RHODOPSIN-I - SPECTROSCOPIC AND FUNCTIONAL-CHARACTERIZATION OF THEMUTATED PROTEINS D76N AND D76A

Both sensory rhodopsin I (SR-I), a phototaxis receptor, and bacteriorh odopsin (BR), a light-driven proton pump, share residues which have be en identified as critical for BR functioning. This includes Asp76, whi ch in the case of bacteriorhodopsin (Asp85) functions both as the Schi ff base counterion and proton acceptor. We found that substituting an Asn for Asp76 (D76N) in SR-I has no effect on its visible absorption u nlike the analogous mutation (D85N) in BR which shifts the absorption to longer wavelengths. The mutated proteins D76N and D76A are also ful ly functional as phototaxis receptors in contrast to BR, where the ana logous substitutions block proton transport. D76N was also found to ex hibit a spectrally normal SR(587)-->S-373 transition. However, FTIR di fference spectroscopy reveals that two bands in the SR(587)-->S-373 di fference spectrum at 1766/1749 cm(-1) (negative/positive), assigned to the C=O stretch mode of a carboxylic acid, disappear in D76N, althoug h no changes are observed in the carboxylate region. In addition, the kinetics and yield of this photoreaction are altered. On this basis, i t is concluded that, unlike Asp85 in bacteriorhodopsin, Asp76 is proto nated in SR-I and undergoes an increase in its hydrogen bonding during the SR(587)-->S-373 transition. This model accounts for the differenc e in color of SR-I and BR and the finding that Asn can substitute for Asp76 without greatly altering the SR-I phenotype. Interestingly, para llels exist between this residue and Asp83 in the visual receptor rhod opsin which has recently been found to exist in a protonated form and to undergo an almost identical change in hydrogen bonding during rhodo psin activation.