Photochemical Properties of the Red‐shifted Channelrhodopsin Chrimson

Color‐tuned variants of channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light‐induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground‐state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state. The photochemical properties of Chrimson, a channelrhodopsin with the longest wavelength absorbance maximum, were characterized at different pH values corresponding to two protonation states of the retinal Schiff base counterion. Femtosecond time‐resolved absorption spectroscopy and flash‐photolysis measurements reveal a strong dependence of Chrimson's photoreaction on the excitation wavelength and pH value. Electrophysiology measurements prove a functional channel opening for both protonation states of the counter‐ion, but via different routes as reflected in the photochemical states observed in spectroscopic experiments.