Origin of the Difference in Proton Transport Direction between Inward and Outward Proton-Pumping Rhodopsins.

Abstract

ConspectusActive transport is a vital and ubiquitous process in biological phenomena. Ion-pumping rhodopsins are light-driven active ion transporters that share a heptahelical transmembrane structural scaffold in which the all-trans retinal chromophore is covalently bonded through a Schiff base to a conserved lysine residue in the seventh transmembrane helix. Bacteriorhodopsin from Halobacterium salinarum was the first ion-pumping rhodopsin to be discovered and was identified as an outward proton-pumping rhodopsin. Since the discovery of bacteriorhodopsin in 1971, many more ion-pumping rhodopsins have been isolated from diverse microorganisms spanning three domains (bacteria, archaea, and eukaryotes) and giant viruses. In addition to proton-pumping rhodopsins, chloride ion- and sodium ion-pumping rhodopsins have also been discovered. Furthermore, diversity of ion-pumping rhodopsins was found in the direction of ion transport; i.e., rhodopsins that pump protons inward have recently been discovered. Very intriguingly, the inward proton-pumping rhodopsins share structural features and many conserved key residues with the outward proton-pumping rhodopsins. However, a central question remains unchanged despite the increasing variety: how and why do the ion-pumping rhodopsins undergo interlocking conformational changes that allow unidirectional ion transfer within proteins? In this regard, it is an effective strategy to compare the structures and their evolutions in the proton-pumping processes of both inward and outward proton-pumping rhodopsins because the comparison sheds light on key elements for the unidirectional proton transport. We elucidated the proton-pumping mechanism of the inward and outward proton-pumping rhodopsins by time-resolved resonance Raman spectroscopy, a powerful technique for tracking the structural evolutions of proteins at work that are otherwise inaccessible.In this Account, we primarily review our endeavors in the elucidation of the proton-pumping mechanisms and determination factors for the transport directions of inward and outward proton-pumping rhodopsins. We begin with a brief summary of previous findings on outward proton-pumping rhodopsins revealed by vibrational spectroscopy. Next, we provide insights into the mechanism of inward proton-pumping rhodopsins, schizorhodopsins, obtained in our studies. Time-resolved resonance Raman spectroscopy provided valuable information about the structures of the retinal chromophore in the unphotolyzed state and intermediates of schizorhodopsins. As we ventured further into our investigations, we succeeded in uncovering the factors determining the directions of proton release and uptake in the retinal Schiff base. While it is intriguing that the proton-pumping rhodopsins actively transport protons against a concentration gradient, it is even more curious that proteins with structural similarities transport protons in opposite directions. Solving the second mystery led to solving the first. When we considered our findings, we realized that we would probably not have been able to elucidate the mechanism if we had studied only the outward pump. Our Account concludes by outlining future opportunities and challenges in the growing research field of ion-pumping rhodopsins, with a particular emphasis on elucidating their sequence-structure-function relationships. We aim to inspire further advances toward the understanding and creation of light-driven active ion transporters.