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, SO₄^2-, 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 SO₄^2-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl^- and SO₄^2- 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 SO₄^2-, and the affinities of Cl^- and SO₄^2- 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 (SO₄^2-) transporter and report its spectroscopic characteristics.

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