Proton Release Group of <i>pharaonis</i> Phoborhodopsin Revealed by ATR-FTIR Spectroscopy

Abstract

The proton release mechanism has been one of the recent interesting topics in the field of microbial rhodopsins since it was established that a protonated water cluster is the proton release group(PRG) in bacteriorhodopsin (BR). pharaonis phoborhodopsin [ppR, also called pharaonis sensory rhodopsinII (pSRII)] is a photoreceptor for negative phototaxis in Natronomonas pharaonis, and in the absence of transducer protein, pHtrII, ppR can pump protons like BR. Fast, BR-like proton release was observed during the lifetime of the M intermediate (ppRM) at low pH, but it was slowed in the absence of Cl-[Iwamoto, M., et al. (2004) Biochemistry 43, 3195]. This observation suggests that Cl- binding controls the pKa of PRG in ppR and ppRM. In this paper, we studied the molecular mechanism of the PRG action in ppR by means of Cl(-)-induced and light-induced difference attenuated total reflection (ATR) FTIRspectroscopy in the aqueous phase. Cl(-)-induced difference ATR-FTIR spectra clearly demonstrated that binding of Cl- to ppR accompanies protonation of a carboxylic acid (C=O stretch at 1724 cm-1). The amino acid was identified as Asp193, because the corresponding band is shifted to 1705 cm-1 in the D193Emutant protein. Light-induced ppRM minus ppR difference ATR-FTIR spectra show the deprotonation signal of Asp193 (at 1724 cm-1) only in the presence of Cl-. The double-difference spectrum between the light-induced changes in the presence and absence of Cl- is a mirror image of the spectrum of Cl binding in the dark, indicating that ppRM formation accompanies deprotonation of Asp193 and dissociation of Cl- simultaneously. It was also shown that structural changes of arginine are involved in these processes by use of [15N]arginine-labeled ppR. We thus conclude that the PRG of ppR includes Asp193, whose pKa changes are controlled by Cl- and Arg72.