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Abstract Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name “mirror proteorhodopsins”, from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest.

Background: Rhodopsins are chromoproteins, which contain retinal, with various necessary functions for energy or photoreception of a cell. Many homologues of rhodopsins were found in the microorganisms of different taxa. Siberian permafrost is a unique biological community, unifying microorganisms adapted to long-term frosts, high osmolarity and lack of light. Exiguobacterium sibiricum is one of the microorganisms that can withstand a wide range of growth conditions. For a long time, the possible role of the found rhodopsin remained unclear in adaptation to extreme environmental conditions. Methods: In this work, we studied complexes of rhodopsin ESR (Exiguobacterium sibiricum rhodopsin - ESR) solubilized in amphipathic polymers (amphipols) A8-35 and PMAL-C12. ESR was expressed in E. coli, purified and inserted in amphipols by standard protocols. The complexes were characterized using the dynamic light scattering method and flash-photolysis, which was the author's designed device for measuring photocycles. Results: Diameters were 4.8 and 5.4 nanometers, all samples were monodisperse and stable for long period. This size indicates the monomericity of the protein. The photocycle of ESR in amphipols was about 150 milliseconds, which was 1.5 times longer than the photocycle of ESR in DDM micelles. This difference could be accounted for by decreasing the conformational mobility of rhodopsin solubilized with into amphipols, rather than detergent micelles. Conclusion: According to this study, the amphipols is a good membrane mimetic for ESR. The protein is stable and functionally active as a light-driven proton pump, which makes it a good candidate for an optogenetic tool. This method could be used for other retinal proteins and lead to the understanding of the physiological necessity of rhodopsin ESR expression by the microorganism E. sibiricum.