Spectroscopic characteristics ofRubricoccus marinusxenorhodopsin (RmXeR) and a putative model for its inward H⁺transport mechanism

Abstract

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 (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29 (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR 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 pK 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 RmXeR 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 RmXeR.

Photochemical Characterization of a New Heliorhodopsin from the Gram-Negative Eubacterium Bellilinea caldifistulae (BcHeR) and Comparison with Heliorhodopsin-48C12

“…The absorption maximum at 550 nm is almost identical to that of HeR-48C12 (Table 1) In many microbial rhodopsins, the all-trans form of the retinal is an active state while the 13-cis form is an inactive state for producing their biological functions. For example, for proton-pumping Rubricoccus marinus xenorhodopsin, 6 the portion of the 13-cis form as the inactive state increases under a high-irradiance environment, probably in order to adaptively reduce the efficiency of the pumping activity. The same senario could be applied to HeR-48C12.…”

Presence of a Haloarchaeal Halorhodopsin-Like Cl⁻ Pump in Marine Bacteria

“…1 ). A recent study showed that RmXeR of SG-29 T functions as a light-driven inward proton pump ( 17 ). R28XeR also belongs to the same cluster of RmXeR, suggesting that the function of R28XeR is an inward H + pump.…”

“…This property is the same as those of the other anion-pumping rhodopsins reported to date ( 11 , 25 , 33 , 35 ). On the other hand, another rhodopsin encoded in the R. marinus SG-29 T gene (RmXeR) showed a light-dependent retinal composition change in the initial state (75 and 45% of all- trans retinal under dark and light conditions, respectively) ( 17 ). The biological significance of RmXeR currently remains unclear.…”

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

“…time-resolved spectroscopy. The apparatus and the procedure for time-resolved flash-photolysis experiments were essentially the same as previously described 34 . In short, transient time-resolved absorption spectra from 375 to 700 nm at 5 nm intervals were measured at time intervals of 0.5 μs using a homemade computer controlled flash photolysis system with a Nd:YAG laser as an actinic light source.…”

“…To monitor proton uptake and release during the photocycle, the pH indicator pyranine (final conc. = 100 μM) was used as previously described 34 . The absorption changes of pyranine were measured using unbuffered samples in a solution containing 0.3 M NaCl, 0.05% DDM to enhance the signals.…”