Structural Evolution\nof Retinal Chromophore in Early\nIntermediates of Inward and Outward Proton-Pumping Rhodopsins

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