Structural Evolution of Retinal Chromophore in Early Intermediates of Inward and Outward Proton-Pumping Rhodopsins.

Proton-pumping rhodopsins, which consist of seven transmembrane helices and have a retinal chromophore bound to a lysine side chain through a Schiff base linkage, offer valuable insights for developing unidirectional ion transporters. Despite identical overall structures and membrane topologies of outward and inward proton-pumping rhodopsins, these proteins transport protons in opposing directions, suggesting a rational mechanism that enables protons to move in different directions within similar protein structures. In the present study, we clarified the chromophore structures in early intermediates of inward and outward proton-pumping rhodopsins. Most importantly, common to both pumps, the hydrogen bond of the Schiff base became stronger in the L intermediate than in the unphotolyzed state. Experimental data on the chromophore structures of the L intermediates and proton-pumping activities indicated that the direction of proton release from the Schiff base during the L-to-M transition is determined not by the structure of the retinal chromophore but by the number of negative charges on the extracellular side of the Schiff base. This is in contrast to the idea that the chromophore configuration is a determinant for the direction of proton uptake. The present study, together with our previous studies, clarifies the determining factors of the transport direction in inward and outward proton-pumping rhodopsins.