The role of retinal chromophore photoisomerization in enhanced inward proton-pumping activity of xenorhodopsin from Nanosalina

Xenorhodopsin (XeR) is the first identified light-driven inward proton pump, exhibiting proton translocation vectoriality opposite to that of bacteriorhodopsin (BR)—a well-characterized outward proton pump rhodopsin. The molecular mechanism governing this vectoriality remains a fundamental question. A distinguishing feature of XeRs is the substitution of the second counterion (Asp212 in BR) with a proline residue located near the protonated retinal Schiff base (PRSB). The absence of a negatively charged residue in XeRs may hinder proton transfer from the Schiff base to the primary counterion (Asp85 in BR), a key determinant of vectoriality. Several studies have reported that XeR from Nanosalina (NsXeR) exhibits higher inward proton-pumping activity than other XeRs, although the underlying molecular mechanism remains unclear. In this study, we analyzed the early photointermediate (K) of NsXeR using light-induced difference Fourier transform infrared spectroscopy, revealing two characteristic features. First, the distinct hydrogen out-of-plane (HOOP) vibrations—indicative of retinal distortion—were absent, suggesting a minimally distorted retinal chromophore post-photoisomerization in NsXeR. Second, the PRSB exhibited a weaker hydrogen bond in the dark state. Interestingly, substituting Pro209 at the second counterion position with alanine or glycine (P209A and P209G) restored HOOP band intensity and strengthened the PRSB hydrogen bond. Importantly, the P209A and P209G mutants demonstrated reduced inward proton-pumping activity and slower recovery in the final thermal isomerization process compared to the wild type. These findings suggest that photoisomerization without retinal distortion enhances inward proton transport in NsXeR.