Vectorial Proton Transport Mechanism of RxR, a Phylogenetically Distinct and Thermally Stable Microbial Rhodopsin

Keiichi Kojima,Tetsuya Ueta,Yuki Sudo,Tomoyasu Noji,Hiroshi Ishikita,Keisuke Saito,Susumu Yoshizawa,Kanae Kanehara

doi:10.1038/s41598-019-57122-2

Keiichi Kojima, Tetsuya Ueta + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-57122-2

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Journal: Scientific Reports	Publication Date: Jan 14, 2020
Citations: 14	License type: open-access

Affiliation: Okayama University, The University of Tokyo

Abstract

Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seven-transmembrane protein that functions as a light-driven proton (H+) pump with the chromophore retinal. To characterize its vectorial proton transport mechanism, mutational and theoretical investigations were performed for carboxylates in the transmembrane region of RxR and the sequential proton transport steps were revealed as follows: (i) a proton of the retinylidene Schiff base (Lys209) is transferred to the counterion Asp74 upon formation of the blue-shifted M-intermediate in collaboration with Asp205, and simultaneously, a respective proton is released from the proton releasing group (Glu187/Glu197) to the extracellular side, (ii) a proton of Asp85 is transferred to the Schiff base during M-decay, (iii) a proton is taken up from the intracellular side to Asp85 during decay of the red-shifted O-intermediate. This ion transport mechanism of RxR provides valuable information to understand other ion transporters since carboxylates are generally essential for their functions.

Highlights

The absorption of light by microbial rhodopsins triggers trans-cis isomerization of the retinal chromophore, leading to structural changes of opsin through a series of distinct photointermediates, such as the K, L, M, Nand O-intermediates[3,8,9]
In 2017, we found a novel proton pump rhodopsin produced by the eubacterium Rubrobacter xylanophilus, which lives in a high temperature environment (i.e., 60 °C)[10]
That rhodopsin, named Rubrobacter xylanophilus rhodopsin (RxR), is a phylogenetically distinct microbial rhodopsin located between the archaeal and eukaryotic proton pumps (Fig. S1A)[10], suggesting that RxR can be an excellent model for understanding the relationships between archaeal, eubacterial and eukaryotic proton pump rhodopsins

Summary

Introduction

The absorption of light by microbial rhodopsins triggers trans-cis isomerization of the retinal chromophore, leading to structural changes of opsin through a series of distinct photointermediates, such as the K-, L-, M-, Nand O-intermediates[3,8,9]. By fitting all the time courses of absorption changes with a triple-exponential function (Fig. S2, gray curves), we estimated the rate constants for the www.nature.com/scientificreports transition processes from the L- to M-intermediate (L → M), from the M- to O-intermediate (M → O), and from the O-intermediate to original state (O → original) at 25, 30, 40, 50 and 60 °C (Table S2).

Results

Conclusion