Molecular Properties of a Novel Inward Proton Pump Rhodopsin, GhXeR

Two kinds of proton pump functions are found in microbial rhodopsins: outward- and inward-directed pumps driven by light. While outward proton pumps convert light energy into proton motive force, the physiological role of light-driven inward proton pumps has not been well understood. In this study, we investigated the molecular properties of a xenorhodopsin (XeR) from the marine bacterium Guptibacillus hwajinpoensis (GhXeR). GhXeR works as an inward proton pump similar to many other XeRs when expressed heterologously in E. coli cells. The proton pump activity per unit time was similar between GhXeR and NsXeR, the known optogenetic tool, whereas GhXeR changed solvent pH for a much longer duration than NsXeR. This suggests that GhXeR is a superior optogenetic tool. Low-temperature UV-visible and FTIR spectroscopy revealed that the primary photoreaction of GhXeR is the retinal photoisomerization from the all-trans to 13-cis form. At 77 K, the chromophore is highly distorted after isomerization, and the peptide backbone structure is not largely changed, suggesting that light energy is stored by the distorted chromophore structure. The protonated Schiff base forms a hydrogen bond in GhXeR, and retinal isomerization weakens the hydrogen bond. Structural changes at 77 K include surrounding side chains such as protonated carboxylic acids and internal water molecules. Since GhXeR is the only rhodopsin in the cultivatable bacterium, GhXeR will be used for understanding the physiological role of the inward proton pump in addition to molecular mechanism studies.