title: The effects of chloride ion binding on the photochemical properties of sensory rhodopsin II from Natronomonas pharaonis.
abstract: 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.
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan. Electronic address: jtamoga@cc.matsuyama-u.ac.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Tamogami
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Iwano
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Matsuyama
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikukawa
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Nara
authors.affiliation: College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Ehime, Japan; Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kamo
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry* / Q000378:metabolism; D055664:Electrochemical Techniques; D006216:Halobacteriaceae / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D007834:Lasers; D011485:Protein Binding; D011522:Protons; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D019843:Archaeal Proteins; D002712:Chlorides; D007477:Ions; D011522:Protons; D025603:Sensory Rhodopsins; D001224:Aspartic Acid