Novel light-driven schizorhodopsins from Antarctic patescibacteria and cyanobacteria

Microbial rhodopsins represent a diverse superfamily of light-sensitive proteins composed of seven transmembrane helices with expanding phylogenetic diversity driven by advances in metagenomics. Among these, schizorhodopsins constitute a divergent family originally identified as inward proton pumps from Promethearchaeota (Asgard archaea). Here, we report that in addition to archaeal schizorhodopsins, many members of the family originate from bacteria and detail a comprehensive biophysical characterization of two schizorhodopsins from uncultured Antarctic bacteria: paSzR from Minisyncoccota (Patescibacteria) and psSzR from a Pseudanabaenacea cyanobacterium. Both proteins function as light-driven inward proton pumps, as confirmed through pH measurements in Escherichia coli cells. Laser-flash photolysis experiments identified multiple photointermediates (K, L, and M) characteristic of microbial rhodopsin photocycles, though with slower turnover rates compared to archaeal schizorhodopsins. Site-directed mutagenesis of conserved residues in the third and sixth transmembrane helices demonstrates differential structural requirements between paSzR and psSzR. Our phylogenetic reconstruction reveals that most bacterial schizorhodopsins cluster in a single lineage distinct from archaeal variants. These findings expand our understanding of microbial rhodopsin diversity and provide crucial insights into alternative molecular mechanisms for light-driven proton translocation, with implications for microbial ecology in extreme environments.