Diverging pH dependence and photocycle dynamics across members of the CryoRhodopsin clade

A new clade of microbial rhodopsins (MRs) called CryoRhodopsins (CryoRs) was recently discovered and characterized to possess a near-UV-absorbing intermediate populated for minutes at neutral to alkaline conditions. A detailed characterization of one member, CryoR1, showed a strong pH dependence, causing a hindered retinal isomerization and a significant acceleration of the photocycle dynamics at acidic conditions. Here, we present a spectroscopic study on the pH dependence of four other CryoRs (CryoR2-5), revealing strongly deviating photophysical properties within this subfamily under acidic conditions. Although all investigated CryoRs possess a minute-long photocycle duration at neutral to alkaline conditions, acidification shortens the photocycle to seconds and leads to variation in the observed photocycle schemes. Furthermore, this is not directly reflected in the absorption spectra and initial photoreactions, since CryoR4 and CryoR5 show very weak pH dependence, whereas CryoR2 and CryoR3 do show pH-dependent shifts. In addition, CryoR3 is unique within both CryoRs and MRs in general, since a near-UV-absorbing species is clearly present throughout the whole pH range in the dark spectrum. The observed diversity suggests strong mechanistic divergence within this clade of MRs, a feature that was not observed for other clades of MRs. It also further supports functional relevance of the long-living near-UV-absorbing state of CryoRs under neutral and alkaline conditions.