RESUMO
Chloride transport by microbial rhodopsins is actively being researched to understand how light energy is converted to drive ion pumping across cell membranes. Chloride pumps have been identified in archaea and eubacteria, and there are similarities and differences in the active site structures between these groups. Thus, it has not been clarified whether a common mechanism underlies the ion pump processes for all chloride-pumping rhodopsins. Here, we applied Raman optical activity (ROA) spectroscopy to two chloride pumps, Nonlabens marinus rhodopsin-3 (NM-R3) and halorhodopsin from the cyanobacterium Mastigocladopsis repens (MrHR). ROA is a vibrational spectroscopy that provides chiral sensitivity, and the sign of ROA signals can reveal twisting of cofactor molecules within proteins. Our ROA analysis revealed that the retinal Schiff base NH group orients toward the C helix and forms a direct hydrogen bond with a nearby chloride ion in NM-R3. In contrast, MrHR is suggested to contain two retinal conformations twisted in opposite directions; one conformation has a hydrogen bond with a chloride ion like NM-R3, while the other forms a hydrogen bond with a water molecule anchored by a G helix residue. These results suggest a general pump mechanism in which the chloride ion is "dragged" by the flipping Schiff base NH group upon photoisomerization.
