Reordering of Retinal Reactions Underlies Reversal of Proton-Pump Directionality

Abstract

Abstract Light-driven proton-pumping rhodopsins transport protons either outward or inward across membranes, but the molecular determinants of transport vectoriality remain unclear. An outward proton-pumping rhodopsin from Pseudomonas putida (PspR) was previously converted into an inward pump by substituting only three amino acid residues. Here, time-resolved resonance Raman spectroscopy reveals that wild-type PspR accepts a proton with a 13-cis retinal chromophore, whereas the engineered inward-pumping PspR triple mutant accepts a proton with an all-trans chromophore, as observed in schizorhodopsins. Intermediate mutant analysis shows that D73F/T78C generates an all-trans M intermediate, while previous transport measurements showed that adding T77S further enhances inward-pumping activity. These findings link reversal of proton-pump vectoriality to reordering of retinal reisomerization and Schiff-base reprotonation, suggesting a strategy for engineering transport directionality in microbial rhodopsins.