Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics.

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 S1-08 strain, 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, K) 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), O, O, and NM-R3'). From these results, we assume that the Cl is released and taken up during the L(N)-O transition from a transiently formed cytoplasmic (CP) binding site and the O-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.