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. While 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, while 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.