title: Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics.
abstract: Several new retinal-based photoreceptor proteins that act as light-driven electrogenic halide ion pumps have recently been discovered. Some of them, called "NTQ" rhodopsins, contain a conserved Asn-Thr-Gln motif in the third or C-helix. In this study, we investigated the photochemical characteristics of an NTQ rhodopsin, Nonlabens marinus rhodopsin 3 (NM-R3), which was discovered in the N. marinus S1-08Tstrain, using static and time-resolved spectroscopic techniques. We demonstrate that NM-R3 binds a Cl-in the vicinity of the retinal chromophore accompanied by a spectral blueshift from 568 nm in the absence of Cl-to 534 nm in the presence of Cl-. From the Cl-concentration dependence, we estimated the affinity (dissociation constant, Kd) for Cl-in the original state as 24 mM, which is ca. 10 times weaker than that of archaeal halorhodopsins but ca. 3 times stronger than that of a marine bacterial Cl-pumping rhodopsin (C1R). NM-R3 showed no dark-light adaptation of the retinal chromophore and predominantly possessed an all-trans-retinal, which is responsible for the light-driven Cl-pump function. Flash-photolysis experiments suggest that NM-R3 passes through five or six photochemically distinct intermediates (K, L(N), O1, O2, and NM-R3'). From these results, we assume that the Cl-is released and taken up during the L(N)-O1transition from a transiently formed cytoplasmic (CP) binding site and the O2-NM-R3' or the NM-R3'-original NM-R3 transitions from the extracellular (EC) side, respectively. We propose a mechanism for the Cl-transport by NM-R3 based on our results and its recently reported crystal structure.
authors.affiliation: Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University , 700-8530 Okayama, Japan.
authors.forename: Takashi
authors.identifier: 0000-0002-6348-6664
authors.initials: T
authors.lastname: Tsukamoto
authors.affiliation: Atmosphere and Ocean Research Institute, The University of Tokyo , Chiba 277-8564, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Yoshizawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikukawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.affiliation: Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University , 700-8530 Okayama, Japan.
authors.forename: Yuki
authors.identifier: 0000-0001-8155-9356
authors.initials: Y
authors.lastname: Sudo
mesh_terms: D002712:Chlorides / Q000378:metabolism*; D041965:Flavobacteriaceae / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D017136:Ion Transport / Q000528:radiation effects; D008027:Light; D008958:Models, Molecular; D012243:Rhodopsin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002712:Chlorides; D012243:Rhodopsin