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

A retinylidene photoreceptor from the cyanobacterium <i>Mastigocladopsis repens</i> (<i>Mr</i>HR) 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 <i>Mr</i>HR + <i>hv</i> → K → L → N1 → N2 → <i>Mr</i>HR' 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 <i>Mr</i>HR. 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 <i>Mr</i>HR.