EFFECTS OF SUBSTITUTIONS D73E, D73N, D103N AND V106M ON SIGNALING ANDPH TITRATION OF SENSORY RHODOPSIN-II

Several mutations in the repellent phototaxis receptor sensory rhodops in II (SRII), in residues homologous to residues important in the rela ted proton pump bacteriorhodopsin, were expressed in Pho81Wr(-), a Hal obacterium salinarum strain deficient in production of SRII and its tr ansducer protein HtrII. The lack of production of SRII and HtrII is sh own to be due to insertion of an ISH2 transposon into the promoter reg ion upstream of the htrII-sopII gene pair. Near wild-type phototaxis r esponses are rescued in Pho81Wr(-) by expression of HtrII with D73E, D 103N or V106M receptors. Partial responses are restored by the HtrII-D 73N pair. From absorption spectroscopy of his-tag-purified receptor pr otein from mutants D73N and D73E we conclude that Asp73 is the primary counterion to the protonated Schiff base in SRII, like the correspond ing Asp85 in bacteriorhodopsin. The absorption maximum of SRII (487 nm ) is shifted to 514 nm in mutant D73N, a 1080 cm(-1) shift identical t o that caused by D85N in bacteriorhodopsin. Acid titration of SRII als o induces the red shift with a pK of 3.0 in wild type. The absorption shift and the pK are nearly the same in V106M and D103N, but the pK is raised to 5.1 in D73E, confirming that Asp73 is the residue responsib le for this spectral transition.