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

Abstract

Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seventransmembrane 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.

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift

“…For protein purification, the E. coli strain BL21 (DE3) was used as a host, as described previously 15 , 38 . The E. coli cells harbouring the plasmid were grown at 37 °C in 2 × YT medium supplemented with ampicillin (final concentration = 50 µg/mL).…”

“…The crude membrane fraction was collected by ultracentrifugation and solubilised with 1.0 w/v % n -dodecyl-β- d -maltoside (DDM). The solubilised-fraction was purified by Ni 2+ affinity column chromatography with a linear gradient of imidazole, as described previously 15 , 38 . The purified protein was concentrated by centrifugation using an Amicon Ultra filter.…”

“…The experiments were performed in buffer (50 mM Tris–HCl (pH 7.0)) containing 300 mM NaCl. To investigate proton uptake and release during the photocycle, we used the pH indicator pyranine (final concentration = 100 µM, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), which has been extensively used to monitor light-induced pH changes in various rhodopsins 3 , 15 , 38 . The pH changes in the bulk environment were measured as the absorption changes of pyranine at 450 nm.…”

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

“…So far, the E. coli cell expression system has been widely utilized for various archaeal and bacterial rhodopsins 9 – 13 , 39 , 41 , 42 . Since Om R2 is phylogenetically distinct from the other characterized eukaryotic rhodopsins, we sought to express the Om R2 recombinant protein using the E. coli cell expression system.…”

“…Finally, we investigated how proton uptake and release happen during the photocycle since Om R2 exhibits a proton pumping function. We measured the flash-induced absorption change at 450 nm of a pH-sensitive fluorochrome, pyranine, which reflects the solvent pH changes as previously described 14 , 42 . As a result, the absorbance of pyranine decreased within 10 ms and then increased within 100 ms.…”