Weak Organic AcidEffect of Bacterial Light-DrivenProton-Pumping Rhodopsin

Microbial rhodopsins are photoreceptor proteins that utilize light to elicit various biological functions. The best-studied microbial rhodopsins are outward proton (H⁺)-pumping rhodopsins, which transport H⁺ from the cytoplasmic to the extracellular side. Recently, the weak organic acid (WOA) effect, specifically the enhancement of pumping activity by WOAs such as acetic acid and indole-3-acetic acid (IAA), was discovered in outward H⁺-pumping rhodopsins from fungi. However, it remains unclear whether the WOA effect exists in nonfungal H⁺-pumping rhodopsins. Here, we revealed that the H⁺-pumping activity of a bacterial outward H⁺ pump rhodopsin, <i>Psp</i>R, from the rhizobacterium Pseudomonas putida, is also enhanced by extracellular acetic acid and IAA. Using transient absorption measurements on purified <i>Psp</i>R protein, we found that extracellular WOAs accelerate cytoplasmic H⁺ uptake and extracellular H⁺ release from a protonated counterion during its photocycle. Furthermore, acetic acid applied on the cytoplasmic side has an inhibitory effect on the H⁺ pump activity of <i>Psp</i>R, which is less significant for IAA and can be mitigated by increasing the H⁺ concentration or introducing a cytoplasmic donor residue. These findings on the WOA effect in a bacterial rhodopsin provide new insights into the physiological function of outward H⁺-pumping rhodopsins in bacteria, particularly in their interaction with plants.