Role of Asp193 in chromophore-protein interaction of pharaonis phoborhodopsin (sensory rhodopsin II)

Pharaonis 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 PK_a of the Schiff base was evaluated. Asp193 corresponds to Glu204 of bacteriorhodopsin (bR). The pK_a 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 pK_a 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 dodecylmaltoside (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.