Sensory rhodopsin II (SRII lmax=487 nm) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum. The SRII receptor is a member of a family of homologous seven-helix proteins that form protonated Schiff base linkages with all-trans-retinal including the ion transporters bacteriorhodopsin (BR lmax=568 nm) and halorhodopsin (HR lmax=578 mn), and the attractant phototaxis receptor sensory rhodopsin I (SRI lmax=587 nm). Several residues which are homologous to residues important in the related proton pump bacteriorhodopsin were mutated by site-directed mutagenesis. Mutated receptor with its transducer HtrII was expressed in Pho81 Wr-, a Halobacterium salinarum strain deficient in the production of SRII and its transducer protein HtrII. The lack of production of SRII and HtrII is shown to be due to insertion of an ISH2 transposon into the promoter region upstream of the htrII-sopII gene pair. Near wild-type phototaxis responses are rescued in Pho81Wr- by expression of HtrII with D73E, D103N V106M or D198N receptors. Partial responses are restored by the HtrII-D73N pair. Photoreaction rate of all mutated receptors is altered. The mutation of valine to methionine in SRII accelerated the decay rate of intermediate (O) approximately 10 times. The V106M pigment exhibits acid and alkaline absorption very similar to those wild-type SRII. The methionine residue is not sufficient to shift the pigment’s absorption to the orange region typical of the other three H. salinarum rhodopsins. From absorption spectroscopy of his-tag-purified receptor protein from mutants D73N and D73E, we conclude that Asp73 is the primary counterion to the protonated Schiff base in SRII, like the corresponding Asp85 in bacteriorhodopsin. The absorption maximum of SRII (487 run) is shifted to 514 run in mutant D73N, a 1080 cm-1 shift identical to that caused by D85N in bacteriohodopsin. Acid titration of SRII also 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, which confirms that Asp73 is the residue responsible for this spectral transition.
Roles of Several Conserved Intramembrane Residues in Signaling and Photochemistry of SRII