Role of Asp193 in Chromophore-Protein Interaction of pharaonis Phoborhodopsin (Sensory Rhodopsin II)

AbstractPharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. By spectroscopic titration of D193N and D193E mutants, the pKa of the Schiff base was evaluated. Asp193 corresponds to Glu204 of bacteriorhodopsin (bR). The pKa of the Schiff base (SBH+) of D193N was ∼10.1–10.0 (at XH+) and ∼11.4–11.6 (at X) depending on the protonation state of a certain residue (designated by X) and independent of Cl−, whereas those of the wild type and D193E were >12. The pKa values of XH+ were ∼11.8–11.2 at the state of SB, 10.5 at SBH+ state in the presence of Cl−, and 9.6 at SBH+ without Cl−. These imply the presence of a long-range interaction in the extracellular channel. Asp193 was suggested to be deprotonated in the present dodecyl-maltoside (DDM) solubilized wild-type ppR, which is contrary to Glu204 of bR. In the absence of salts, the irreversible denaturation of D193N (but not the wild type and D193E) occurred via a metastable state, into which the addition of Cl− reversed the intact pigment. This suggests that the negative charge at residue 193, which can be substituted by Cl−, is necessary to maintain the proper conformation in the DDM-solubilized ppR