Mutations conferring SO(4)(2−) pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant

Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl(-) and Br(-), but SyHR also transports SO(4)(2−), despite the close sequence similarity to MrHR. We sought a determinant that could confer SO(4)(2−) pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO(4)(2−) transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO(4)(2−) pump activity. Notably, the MrHR and SyHR mutants did not show SO(4)(2−) induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO(4)(2−) in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO(4)(2−) into their centers after illumination and release it before the ends of the photoreactions.