Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

Department of Aquatic Microbial Ecology Institute of Hydrobiology Biology Centre of the Academy of Sciences of the Czech Republic České Budějovice Czech Republic

Department of Life Science and Applied Chemistry Nagoya Institute of Technology Showa ku Nagoya 466 8555 Japan

Department of Molecular Biology and Biotechnology Faculty of Biology and Geology Babeş Bolyai University Cluj Napoca Romania

Department of Physics Nagoya University Nagoya 464 8602 Japan

Exploratory Research Center on Life and Living Systems Higashiyama Myodaiji Okazaki Aichi 444 8787 Japan

Faculty of Biology Technion Israel Institute of Technology Haifa Israel

OptoBioTechnology Research Center Nagoya Institute of Technology Showa ku Nagoya 466 8555 Japan

PRESTO Japan Science and Technology Agency 4 1 8 Honcho Kawaguchi Saitama 332 0012 Japan

The Institute for Solid State Physics The University of Tokyo 5 1 5 Kashiwanoha Kashiwa Chiba 277 8581 Japan

See more in PubMed

Ernst O. P., Lodowski D. T., Elstner M., Hegemann P., Brown L. S., Kandori H., Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms. Chem. Rev. 114, 126–163 (2014). PubMed PMC

Philosof A., Béjà O., Bacterial, archaeal and viral-like rhodopsins from the Red Sea. Environ. Microbiol. Rep. 5, 475–482 (2013). PubMed

Mukherjee S., Hegemann P., Broser M., Enzymerhodopsins: Novel photoregulated catalysts for optogenetics. Curr. Opin. Struct. Biol. 57, 118–126 (2019). PubMed

Govorunova E. G., Sineshchekov O. A., Hemmati R., Janz R., Morelle O., Melkonian M., Wong G. K.-S., Spudich J. L., Extending the time domain of neuronal silencing with cryptophyte anion channelrhodopsins. eNeuro 5, ENEURO.0174-18.2018 (2018). PubMed PMC

Pushkarev A., Inoue K., Larom S., Flores-Uribe J., Singh M., Konno M., Tomida S., Ito S., Nakamura R., Tsunoda S. P., Philosof A., Sharon I., Yutin N., Koonin E. V., Kandori H., Béjà O., A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. Nature 558, 595–599 (2018). PubMed PMC

Spang A., Saw J. H., Jørgensen S. L., Zaremba-Niedzwiedzka K., Martijn J., Lind A. E., van Eijk R., Schleper C., Guy L., Ettema T. J. G., Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature 521, 173–179 (2015). PubMed PMC

Spang A., Eme L., Saw J. H., Caceres E. F., Zaremba-Niedzwiedzka K., Lombard J., Guy L., Ettema T. J. G., Asgard archaea are the closest prokaryotic relatives of eukaryotes. PLoS Genet. 14, e1007080 (2018). PubMed PMC

Bulzu P.-A., Andrei A.-Ş., Salcher M. M., Mehrshad M., Inoue K., Kandori H., Béjà O., Ghai R., Banciu H. L., Casting light on Asgardarchaeota metabolism in a sunlit microoxic niche. Nat. Microbiol. 4, 1129–1137 (2019). PubMed

Inoue K., Ito S., Kato Y., Nomura Y., Shibata M., Uchihashi T., Tsunoda S. P., Kandori H., A natural light-driven inward proton pump. Nat. Commun. 7, 13415 (2016). PubMed PMC

Shevchenko V., Mager T., Kovalev K., Polovinkin V., Alekseev A., Juettner J., Chizhov I., Bamann C., Vavourakis C., Ghai R., Gushchin I., Borshchevskiy V., Rogachev A., Melnikov I., Popov A., Balandin T., Rodriguez-Valera F., Manstein D. J., Bueldt G., Bamberg E., Gordeliy V., Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach. Sci. Adv. 3, e1603187 (2017). PubMed PMC

Inoue S., Yoshizawa S., Nakajima Y., Kojima K., Tsukamoto T., Kikukawa T., Sudo Y., Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism. Phys. Chem. Chem. Phys. 20, 3172–3183 (2018). PubMed

Sunagawa S., Coelho L. P., Chaffron S., Kultima J. R., Labadie K., Salazar G., Djahanschiri B., Zeller G., Mende D. R., Alberti A., Cornejo-Castillo F. M., Costea P. I., Cruaud C., d'Ovidio F., Engelen S., Ferrera I., Gasol J. M., Guidi L., Hildebrand F., Kokoszka F., Lepoivre C., Lima-Mendez G., Poulain J., Poulos B. T., Royo-Llonch M., Sarmento H., Vieira-Silva S., Dimier C., Picheral M., Searson S., Kandels-Lewis S.; Tara Oceans coordinators, Bowler C., de Vargas C., Gorsky G., Grimsley N., Hingamp P., Iudicone D., Jaillon O., Not F., Ogata H., Pesant S., Speich S., Stemmann L., Sullivan M. B., Weissenbach J., Wincker P., Karsenti E., Raes J., Acinas S. G., Bork P., Structure and function of the global ocean microbiome. Science 348, 1261359 (2015). PubMed

Brum J. R., Ignacio-Espinoza J. C., Roux S., Doulcier G., Acinas S. G., Alberti A., Chaffron S., Cruaud C., de Vargas C., Gasol J. M., Gorsky G., Gregory A. C., Guidi L., Hingamp P., Iudicone D., Not F., Ogata H., Pesant S., Poulos B. T., Schwenck S. M., Speich S., Dimier C., Kandels-Lewis S., Picheral M., Searson S.; Tara Oceans Coordinators, Bork P., Bowler C., Sunagawa S., Wincker P., Karsenti E., Sullivan M. B., Patterns and ecological drivers of ocean viral communities. Science 348, 1261498 (2015). PubMed

Hingamp P., Grimsley N., Acinas S. G., Clerissi C., Subirana L., Poulain J., Ferrera I., Sarmento H., Villar E., Lima-Mendez G., Faust K., Sunagawa S., Claverie J.-M., Moreau H., Desdevises Y., Bork P., Raes J., de Vargas C., Karsenti E., Kandels-Lewis S., Jaillon O., Not F., Pesant S., Wincker P., Ogata H., Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes. ISME J. 7, 1678–1695 (2013). PubMed PMC

Philosof A., Yutin N., Flores-Uribe J., Sharon I., Koonin E. V., Béjà O., Novel abundant oceanic viruses of uncultured marine group II euryarchaeota. Curr. Biol. 27, 1362–1368 (2017). PubMed PMC

Nack M., Radu I., Gossing M., Bamann C., Bamberg E., von Mollard G. F., Heberle J., The DC gate in Channelrhodopsin-2: Crucial hydrogen bonding interaction between C128 and D156. Photochem. Photobiol. Sci. 9, 194–198 (2010). PubMed

Scheib U., Stehfest K., Gee C. E., Körschen H. G., Fudim R., Oertner T. G., Hegemann P., The rhodopsin–guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling. Sci. Signal. 8, rs8 (2015). PubMed

Watari M., Ikuta T., Yamada D., Shihoya W., Yoshida K., Tsunoda S. P., Nureki O., Kandori H., Spectroscopic study of the transmembrane domain of a rhodopsin–phosphodiesterase fusion protein from a unicellular eukaryote. J. Biol. Chem. 294, 3432–3443 (2019). PubMed PMC

Inoue K., Kato Y., Kandori H., Light-driven ion-translocating rhodopsins in marine bacteria. Trends Microbiol. 23, 91–98 (2014). PubMed

Inoue K., Tsukamoto T., Shimono K., Suzuki Y., Miyauchi S., Hayashi S., Kandori H., Sudo Y., Converting a light-driven proton pump into a light-gated proton channel. J. Am. Chem. Soc. 137, 3291–3299 (2015). PubMed

Fudim R., Szczepek M., Vierock J., Vogt A., Schmidt A., Kleinau G., Fischer P., Bartl F., Scheerer P., Hegemann P., Design of a light-gated proton channel based on the crystal structure of Coccomyxa rhodopsin. Sci. Signal. 12, eaav4203 (2019). PubMed

Shibata M., Inoue K., Ikeda K., Konno M., Singh M., Kataoka C., Abe-Yoshizumi R., Kandori H., Uchihashi T., Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy. Sci. Rep. 8, 8262 (2018). PubMed PMC

Druckmann S., Ottolenghi M., Pande A., Pande J., Callender R. H., Acid-base equilibrium of the Schiff base in bacteriorhodopsin. Biochemistry 21, 4953–4959 (1982). PubMed

Inoue K., Tahara S., Kato Y., Takeuchi S., Tahara T., Kandori H., Spectroscopic study of proton-transfer mechanism of inward proton-pump rhodopsin, Parvularcula oceani xenorhodopsin. J. Phys. Chem. B 122, 6453–6461 (2018). PubMed

Aton B., Doukas A. G., Callender R. H., Becher B., Ebrey T. G., Resonance Raman studies of the purple membrane. Biochemistry 16, 2995–2999 (1977). PubMed

Rothschild K. J., Marrero H., Braiman M., Mathies R., Primary photochemistry of bacteriorhodopsin: Comparison of Fourier transform infrared difference spectra with resonance Raman spectra. Photochem. Photobiol. 40, 675–679 (1984). PubMed

Lórenz-Fonfría V. A., Muders V., Schlesinger R., Heberle J., Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1. J. Chem. Phys. 141, 22D507 (2014). PubMed

Ito S., Kato H. E., Taniguchi R., Iwata T., Nureki O., Kandori H., Water-containing hydrogen-bonding network in the active center of channelrhodopsin. J. Am. Chem. Soc. 136, 3475–3482 (2014). PubMed

Muroda K., Nakashima K., Shibata M., Demura M., Kandori H., Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps. Biochemistry 51, 4677–4684 (2012). PubMed

Ito S., Sugita S., Inoue K., Kandori H., FTIR analysis of a light-driven inward proton-pumping rhodopsin at 77 K. Photochem. Photobiol. 93, 1381–1387 (2017). PubMed

Petrovskaya L. E., Lukashev E. P., Chupin V. V., Sychev S. V., Lyukmanova E. N., Kryukova E. A., Ziganshin R. H., Spirina E. V., Rivkina E. M., Khatypov R. A., Erokhina L. G., Gilichinsky D. A., Shuvalov V. A., Kirpichnikov M. P., Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump. FEBS Lett. 584, 4193–4196 (2010). PubMed

Kawanabe A., Furutani Y., Jung K.-H., Kandori H., Engineering an inward proton transport from a bacterial sensor rhodopsin. J. Am. Chem. Soc. 131, 16439–16444 (2009). PubMed

Doig S. J., Reid P. J., Mathies R. A., Picosecond time-resolved resonance Raman spectroscopy of bacteriorhodopdn's J, K, and KL intermediates. J. Phys. Chem. 95, 6372–6379 (1991).

Garczarek F., Gerwert K., Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy. Nature 439, 109–112 (2006). PubMed

Gerwert K., Freier E., Wolf S., The role of protein-bound water molecules in microbial rhodopsins. Biochim. Biophys. Acta 1837, 606–613 (2014). PubMed

Lórenz-Fonfría V. A., Kandori H., Spectroscopic and kinetic evidence on how bacteriorhodopsin accomplishes vectorial proton transport under functional conditions. J. Am. Chem. Soc. 131, 5891–5901 (2009). PubMed

Nango E., Royant A., Kubo M., Nakane T., Wickstrand C., Kimura T., Tanaka T., Tono K., Song C., Tanaka R., Arima T., Yamashita A., Kobayashi J., Hosaka T., Mizohata E., Nogly P., Sugahara M., Nam D., Nomura T., Shimamura T., Im D., Fujiwara T., Yamanaka Y., Jeon B., Nishizawa T., Oda K., Fukuda M., Andersson R., Båth P., Dods R., Davidsson J., Matsuoka S., Kawatake S., Murata M., Nureki O., Owada S., Kameshima T., Hatsui T., Joti Y., Schertler G., Yabashi M., Bondar A.-N., Standfuss J., Neutze R., Iwata S., A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354, 1552–1557 (2016). PubMed

Balashov S. P., Govindjee R., Kono M., Imasheva E., Lukashev E., Ebrey T. G., Crouch R. K., Menick D. R., Feng Y., Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle. Biochemistry 32, 10331–10343 (1993). PubMed

Inoue K., Ono H., Abe-Yoshizumi R., Yoshizawa S., Ito H., Kogure K., Kandori H., A light-driven sodium ion pump in marine bacteria. Nat. Commun. 4, 1678 (2013). PubMed

Ikeura Y., Shimono K., Iwamoto M., Sudo Y., Kamo N., Arg-72 of pharaonis phoborhodopsin (sensory rhodopsin II) is important for the maintenance of the protein structure in the solubilized states. Photochem. Photobiol. 77, 96–100 (2003). PubMed

Tanimoto T., Shibata M., Belenky M., Herzfeld J., Kandori H., Altered hydrogen bonding of Arg82 during the proton pump cycle of bacteriorhodopsin: A low-temperature polarized FTIR spectroscopic study. Biochemistry 43, 9439–9447 (2004). PubMed

Wood J. N., Bevan S. J., Coote P. R., Dunn P. M., Harmar A., Hogan P., Latchman D. S., Morrison C., Rougon G., Theveniau M., Wheatley S., Novel cell lines display properties of nociceptive sensory neurons. Proc. Biol. Sci. 241, 187–194 (1990). PubMed

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J.-Y., White D. J., Hartenstein V., Eliceiri K., Tomancak P., Cardona A., Fiji: An open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012). PubMed PMC

Bayburt T. H., Grinkova Y. V., Sligar S. G., Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett. 2, 853–856 (2002).

Uchihashi T., Kodera N., Ando T., Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy. Nat. Protoc. 7, 1193–1206 (2012). PubMed

Structural insights into light harvesting by antenna-containing rhodopsins in marine Asgard archaea

The Evolutionary Kaleidoscope of Rhodopsins

Structure-based insights into evolution of rhodopsins