Cryo-Raman Observation on the Photocycle of a Light-Driven Cl– Pump from Mastigocladopsis repens at High Cl– Concentration

A retinylidene photoreceptor from the cyanobacterium Mastigocladopsis repens (MrHR) is a novel type of light-driven Cl^– pump that has a structural similarity to the archaeal H⁺ pumps but transports Cl^–. An open question regarding the photocycle of this photoreceptor involves the role of a late red-shifted photoproduct, the O intermediate, which is reportedly present at high Cl^– concentrations. In this study, we used cryo-Raman spectroscopy to examine the chromophore structures of the photointermediates at a high Cl^– concentration. When compared to the photointermediates formed as MrHR + hv → K → L → N1 → N2 → MrHR′ at a low Cl^– concentration, we found that N2 kinetically disappears at a high Cl^– concentration. This indicated that N2 is the transient Cl^–-unbound state of MrHR. In addition, we found that no Raman signal of the O intermediate is observed at the high Cl^– concentration, while the signal from N1 is exclusively observed in the later stages of the photocycle. After confirming that N1’s absorption spectrum displays an extending red edge, we concluded that the O intermediate is attributable to the red edge of the absorption band of N1. Here, we report a revised understanding of the photocycle for Cl^– transport of MrHR.