Roles of basic amino acid residues in substrate binding and transport of the light-driven anion pump Synechocystis halorhodopsin (SyHR).

Microbial rhodopsins are photoreceptive seven-transmembrane α-helical proteins, many of which function as ion transporters, primarily for small monovalent ions such as Na, K, Cl, Br, and I. Synechocystis halorhodopsin (SyHR), identified from the cyanobacterium Synechocystis sp. PCC 7509, uniquely transports the polyatomic divalent SO inward, in addition to monovalent anions (Cl and Br). In this study, we conducted alanine-scanning mutagenesis on twelve basic amino acid residues to investigate the anion transport mechanism of SyHR. We quantitatively evaluated the Cl and SO transport activities of the WT SyHR and its mutants. The results showed a strong correlation between the Cl and SO transport activities among them (R = 0.94), suggesting a shared pathway for both anions. Notably, the R71A mutation selectively abolished SO transport activity while maintaining Cl transport, whereas the H167A mutation significantly impaired both Cl and SO transport. Furthermore, spectroscopic analysis revealed that the R71A mutant lost its ability to bind SO due to the absence of a positive charge, while the H167A mutant failed to accumulate the O intermediate during the photoreaction cycle (photocycle) due to reduced hydrophilicity. Additionally, computational analysis revealed the SO binding modes and clarified the roles of residues involved in its binding around the retinal chromophore. Based on these findings and previous structural information, we propose that the positive charge and hydrophilicity of Arg71 and His167 are crucial for the formation of the characteristic initial and transient anion-binding site of SyHR, enabling its unique ability to bind and transport both Cl and SO.