Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism"Complex molecular systems; supramolecules, biomolecules and interfaces" themed issue of PCCP.Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cp05033j

• Main Authors: Inoue, Saki, Yoshizawa, Susumu, Nakajima, Yu, Kojima, Keiichi, Tsukamoto, Takashi, Kikukawa, Takashi, Sudo, Yuki
• Format: Journal Article
• Language: English
• Published: 31.01.2018
• ISSN: 1463-9076; 1463-9084
• DOI: 10.1039/c7cp05033j

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani ( Po XeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29 T ( Rm XeR). Escherichia coli cells expressing recombinant Rm XeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that Rm XeR is a light-driven "inward" proton pump as is Po XeR. The spectroscopic properties of purified Rm XeR were investigated and compared with those of Po XeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum . Action spectroscopy revealed that Rm XeR with all- trans retinal is responsible for the light-driven inward proton transport activity, but not with 13- cis retinal. From pH titration experiments and mutational analysis, we estimated the p K a values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for Rm XeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of Rm XeR. On the basis of functional and spectroscopic characterization, we propose a model for the inward proton transport in Rm XeR, a newly discovered microbial rhodopsin.