Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity

Chloride transport by microbial rhodopsins is activelybeing researchedto understand how light energy is converted to drive ion pumping acrosscell membranes. Chloride pumps have been identified in archaea andeubacteria, and there are similarities and differences in the activesite structures between these groups. Thus, it has not been clarifiedwhether a common mechanism underlies the ion pump processes for allchloride-pumping rhodopsins. Here, we applied Raman optical activity(ROA) spectroscopy to two chloride pumps, Nonlabensmarinus rhodopsin-3 (NM-R3) and halorhodopsin fromthe cyanobacterium Mastigocladopsis repens (MrHR). ROA is a vibrational spectroscopy that provides chiral sensitivity,and the sign of ROA signals can reveal twisting of cofactor moleculeswithin proteins. Our ROA analysis revealed that the retinal Schiffbase NH group orients toward the C helix and forms a direct hydrogenbond with a nearby chloride ion in NM-R3. In contrast, MrHR is suggestedto 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 anchoredby a G helix residue. These results suggest a general pump mechanismin which the chloride ion is dragged by the flippingSchiff base NH group upon photoisomerization.

Automated summary

Research on microbial rhodopsins is being conducted to understand how light energy is converted into ion pumping across cell membranes. Chloride pumps have been found in archaea and eubacteria, but it is unclear if there is a common mechanism for all chloride-pumping rhodopsins. Using Raman optical activity spectroscopy, two chloride pumps, Nonlabensmarinus rhodopsin-3 (NM-R3) and halorhodopsin from the cyanobacterium Mastigocladopsis repens (MrHR), were analyzed. Results showed differences in the hydrogen bonding and conformations of retinal Schiff base, suggesting a general mechanism where chloride ions are dragged by the flipping Schiff base NH group upon photoisomerization.