Demonstration of a Light-Driven SO₄^2– Transporter and Its Spectroscopic Characteristics

Abstract

In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H, Na, and Cl. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl or SO. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl and SO with a subsequent secondary inward H movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl and from 542 to 556 nm for SO, and the affinities of Cl and SO were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO) transporter and report its spectroscopic characteristics.

Molecular details of the unique mechanism of chloride transport by a cyanobacterial rhodopsin

“…40,43 Finally, the most recent addition to the ranks of anion transporting microbial rhodopsins is a distinct group of cyanobacterial light-driven anion pumps with the TSD motif. 44,45 It should be noted that the motif-forming Asp is not strictly conserved and can be present as Val, Leu, or Ile in some members of the group (Fig. S1, ESI †).…”

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

“…Overall, four of the 40 rhodopsins showed red-shifted absorption > 20 nm compared with the base wavelengths (Table 1): three were halorhodopsins (HRs) from bacterial species 9,30,31 (to distinguish classical HRs from archaeal species, these are hereafter referred to as bacterial-halorhodopsins [BacHRs]), and one was a PR 32 . Their ion-transport activities were then investigated by expressing in E. coli cells and observing the pH change in external solvent (Fig.…”

“…Upon light illumination, BacHRs from Rubrivirga marina and Myxosarcina sp. GI1 showed significant alkalization of external solvent, which was enhanced by addition of the protonophore (CCCP), which increases the H + permeability of the cell membrane, and the lightdependent alkalizations disappeared when anions were exchanged from Cl − to SO 4 2− , indicating that these were light-driven Cl − pumps, similar to other rhodopsins in the same BacHR subfamily 9,30 . By contrast, Cyanothece sp.…”

“…1b). Ion-transporting rhodopsins can transport several types of cations and anions, including H + , Na + , K + , halides (Cl − , Br − , I − ), NO − , and SO4 2–1,7–9 . The molecular mechanisms of ion-transporting rhodopsins have been detailed in numerous biophysical, structural, and theoretical studies 1 .…”

Conversion of microbial rhodopsins: insights into functionally essential elements and rational protein engineering

“…Recently, a new class of Cl − pump, which has a TSD-motif, was reported from cyanobacterium Mastigocladopsis repens (MrHR) (Hasemi et al 2016) and Synechocystis sp. PCC 7509 (SyHR) (Niho et al 2017). This pump retains the aspartic acid homologous to the H + donor of archeal H + pumps (Asp96 in HsBR).…”