Calcium-Sensitive Microbial Rhodopsin VirChR1: A Femtosecond to Second Photocycle Study

Viral rhodopsins are light-gated cation channels representing a novel class of microbial rhodopsins. For viral rhodopsin 1 subfamily members VirChR1 and OLPVR1, channel activity is abolished above a certain calcium concentration. Here we present a calcium-dependent spectroscopic analysis of VirChR1 on the femtosecond to second time scale. Unlike channelrhodopsin-2, VirChR1 possesses two intermediate states P₁ and P₂ on the ultrafast time scale, similar to J and K in ion-pumping rhodopsins. Subsequently, we observe multifaceted photocycle kinetics with up to seven intermediate states. Calcium predominantly affects the last photocycle steps, including the appearance of additional intermediates P₆^Ca and P₇ representing the blocked channel. Furthermore, the photocycle of the counterion variant D80N is drastically altered, yielding intermediates with different spectra and kinetics compared to those of the wt. These findings demonstrate the central role of the counterion within the defined reaction sequence of microbial rhodopsins that ultimately defines the protein function.