The effects of chloride ion binding on the photochemical properties of sensory rhodopsin II from Natronomonas pharaonis

• Cl− binds to NpSRII, even in detergent.
• The binding of Cl− to NpSRII depends on the pH of the solvent.
• Cl− binding induces several changes in the photochemical properties of NpSRII.
• Cl− binding allows the initial proton release from Asp193 through its proper pKa control.
• The Cl− binding site is assigned to the area near Arg72 and Asp193.

Whether Cl− binds to the sensory rhodopsin II from Natronomonas pharaonis (NpSRII) that acts as a negative phototaxis receptor remains controversial. Two previous photoelectrochemical studies using SnO2 transparent electrodes and ATR–FTIR demonstrated that Cl− binding affects the photoinduced proton release from Asp193 in phospholipid (PC)-reconstituted NpSRII (Iwamoto et al., 2004; Kitade et al., 2009). In this study, we investigated the effects of Cl− on the photochemistry of NpSRII solubilized by detergent (DDM). Even under these conditions, Cl− could bind to NpSRII with a Kd of approximately 250 mM; this value is ∼10-fold larger than that in the PC membrane. The binding of Cl− to NpSRII depended on the pH of the medium. In addition, Cl− binding induced the following effects: (1) a small red shift in the absorbance spectrum originating from the partial protonation of Asp75, (2) the formation of an interaction through a hydrogen-bonding network between Asp75 and Asp193, which is a proton-releasing residue, (3) several changes of the kinetic behavior of the photocycle, and (4) a photoinduced initial proton release from Asp193. The pKa values of Asp193 at various Cl− concentrations were also estimated. Based on the difference between the pKa values of Asp193 in Cl− bound and unbound NpSRII, the distance between the bound Cl− and Asp193 was determined to be approximately 6.1 Å, which agrees with the value estimated from the crystal structure presented by Royant et al. (2001). Therefore, the Cl− binding site affecting the photochemical properties of NpSRII is identical to the site proposed by Royant et al. (2001). This assignment was also supported by an experiment that introduced a mutation at Arg72.

Figure optionsDownload as PowerPoint slide