Krokinobacter eikastus rhodopsin 2 (KR2) is a microbial light-driven ion pump that transports Na+ out of the cell. The active transport is coupled to a photocycle of the retinal chromophore, covalently attached to the protein via a Schiff base linkage. In this work, we have employed time-resolved pump-probe resonance Raman (TR RR) spectroscopy to characterize the operational switches of KR2 that control Na+ uptake from the cytosol and release to the exterior. The analysis was based on a comprehensive spectroscopic investigation of the parent state using different excitation lines. Two substates were identified in which the Asp116 counterion and water positions differ with respect to the Schiff base. In line with published crystallographic data, one of the substates was ascribed to the active configuration that binds Na+ after deprotonation of the Schiff base. The main advantage of the present TR RR method is that chromophore structures of the photocycle intermediates and their kinetics can be determined. Thus, we identified two redshifted intermediates O1 and O2 formed within ca. 2 and 5-7 ms after illumination, in which the chromophore adopts a protonated 13-cis and all-trans configuration, respectively. This isomerization is ascribed to be the switch for Na+ release.
Schafer et al. (Thu,) studied this question.
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