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

J Phys Chem B. 2017 Mar 9;121(9):2027-2038. doi: 10.1021/acs.jpcb.6b11101. Epub 2017 Mar 1.

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 S1-08T 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, 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)-O1 transition 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chlorides / metabolism*
  • Flavobacteriaceae / chemistry*
  • Flavobacteriaceae / metabolism
  • Flavobacteriaceae / radiation effects*
  • Ion Transport / radiation effects
  • Light*
  • Models, Molecular
  • Rhodopsin / chemistry*
  • Rhodopsin / isolation & purification
  • Rhodopsin / metabolism*

Substances

  • Chlorides
  • Rhodopsin