Despite extensive research on carotenoids and microbial rhodopsins in aquatic environments, a fundamental understanding of the binding requirements of carotenoids that serve as auxiliary light-harvesting antennas for rhodopsins is still lacking. Our recent discovery of 3-hydroxylated xanthophyll-binding proteorhodopsins and xanthorhodopsins prompted us to investigate the role of keto and hydroxy functional groups in carotenoid binding to rhodopsins and their influence on energy transfer to the retinal chromophore. In this study, we examined the binding of 12 carotenoids to rhodopsin Kin4B8 (a protein of the xanthorhodopsin family, GenBank: OP056329) and assessed the energy transfer between the carotenoid and the retinal chromophore. We found that 3-hydroxylated xanthophylls were the most effective light-harvesting antennas among the carotenoids studied. While 4-ketocarotenoids also bound to the protein, their energy transfer efficiency was significantly reduced. In contrast, the presence of a 4-hydroxy group or the substitution of the β-ionone ring by an ε-ionone ring completely prevented binding. Furthermore, mutagenesis studies of Kin4B8 suggest that specific residues play a key role in the selective binding of carotenoids. These findings provide valuable insights into the structural determinants of rhodopsin-carotenoid interactions, which may aid in predicting the recruitment of various carotenoid antennas by retinal proteins.

Centre Algatech Institute of Microbiology Novohradská Třeboň 37981 Czech Republic

Department of Molecular Chemistry and Materials Science Weizmann Institute of Science Rehovot 7610001 Israel

Faculty of Biology Technion Israel Institute of Technology Haifa 3200003 Israel

Faculty of Science University of South Bohemia Branišovská 1760 370 05 České Budějovice Czech Republic

Fritz Haber Center for Molecular Dynamics Research Institute of Chemistry The Hebrew University of Jerusalem Jerusalem 9190401 Israel

Laboratory of Biochemistry and Molecular Biology Faculty of Experimental Sciences Marine International Campus of Excellence University of Huelva Huelva 21071 Spain

The Nancy and Stephen Grand Technion Energy Program Technion Israel Institute of Technology Haifa 3200003 Israel

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Beja O., Aravind L., Koonin E. V., Suzuki M. T., Hadd A., Nguyen L. P., Jovanovich S. B., Gates C. M., Feldman R. A., Spudich J. L.. et al. Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. Science. 2000;289(5486):1902–1906. doi: 10.1126/science.289.5486.1902. PubMed DOI

Béjà O., Spudich E. N., Spudich J. L., Leclerc M., DeLong E. F.. Proteorhodopsin phototrophy in the ocean. Nature. 2001;411(6839):786–789. doi: 10.1038/35081051. PubMed DOI

Finkel O. M., Beja O., Belkin S.. Global abundance of microbial rhodopsins. ISME J. 2013;7(2):448–451. doi: 10.1038/ismej.2012.112. PubMed DOI PMC

Boichenko V. A., Wang J. M., Anton J., Lanyi J. K., Balashov S. P.. Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration. Biochim. Biophys. Acta. 2006;1757(12):1649–1656. doi: 10.1016/j.bbabio.2006.08.012. PubMed DOI PMC

Balashov S. P., Imasheva E. S., Boichenko V. A., Anton J., Wang J. M., Lanyi J. K.. Xanthorhodopsin: A proton pump with a light-harvesting carotenoid antenna. Science. 2005;309(5743):2061–2064. doi: 10.1126/science.1118046. PubMed DOI PMC

Oesterhelt D., Stoeckenius W.. Functions of a new photoreceptor membrane. Proc. Natl. Acad. Sci. U.S.A. 1973;70(10):2853–2857. doi: 10.1073/pnas.70.10.2853. PubMed DOI PMC

Balashov S. P., Imasheva E. S., Wang J. M., Lanyi J. K.. Excitation energy-transfer and the relative orientation of retinal and carotenoid in xanthorhodopsin. Biophys. J. 2008;95(5):2402–2414. doi: 10.1529/biophysj.108.132175. PubMed DOI PMC

Luecke H., Schobert B., Stagno J., Imasheva E. S., Wang J. M., Balashov S. P., Lanyi J. K.. Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore. Proc. Natl. Acad. Sci. U.S.A. 2008;105(43):16561–16565. doi: 10.1073/pnas.0807162105. PubMed DOI PMC

Balashov S. P., Imasheva E. S., Choi A. R., Jung K. H., Liaaen-Jensen S., Lanyi J. K.. Reconstitution of gloeobacter rhodopsin with echinenone: Role of the 4-keto group. Biochemistry. 2010;49(45):9792–9799. doi: 10.1021/bi1014166. PubMed DOI PMC

Imasheva E. S., Balashov S. P., Choi A. R., Jung K. H., Lanyi J. K.. Reconstitution of gloeobacter violaceus rhodopsin with a light-harvesting carotenoid antenna. Biochemistry. 2009;48(46):10948–10955. doi: 10.1021/bi901552x. PubMed DOI PMC

Iyer E. S., Gdor I., Eliash T., Sheves M., Ruhman S.. Efficient femtosecond energy transfer from carotenoid to retinal in gloeobacter rhodopsin-salinixanthin complex. J. Phys. Chem. B. 2015;119(6):2345–2349. doi: 10.1021/jp506639w. PubMed DOI

Chuon K., Shim J. G., Kim S. H., Cho S. G., Meas S., Kang K. W., Kim J. H., Das I., Sheves M., Jung K. H.. The role of carotenoids in proton-pumping rhodopsin as a primitive solar energy conversion system. J. Photochem. Photobiol., B. 2021;221:112241. doi: 10.1016/j.jphotobiol.2021.112241. PubMed DOI

Chuon K., Shim J. G., Kang K. W., Cho S. G., Hour C., Meas S., Kim J. H., Choi A., Jung K. H.. Carotenoid binding in gloeobacteria rhodopsin provides insights into divergent evolution of xanthorhodopsin types. Commun. Biol. 2022;5(1):512. doi: 10.1038/s42003-022-03429-2. PubMed DOI PMC

Shim J.-G., Choun K., Kang K.-W., Kim J.-H., Cho S.-G., Jung K.-H.. The binding of secondary chromophore for thermally stable rhodopsin makes more stable with temperature. Protein Sci. 2022;31(9):e4386. doi: 10.1002/pro.4386. DOI

Anashkin V. A., Bertsova Y. V., Mamedov A. M., Mamedov M. D., Arutyunyan A. M., Baykov A. A., Bogachev A. V.. Engineering a carotenoid-binding site in dokdonia sp. PRO95 Na+-translocating rhodopsin by a single amino acid substitution. Photosynth. Res. 2018;136(2):161–169. doi: 10.1007/s11120-017-0453-0. PubMed DOI

Ghosh M., Misra R., Bhattacharya S., Majhi K., Jung K. H., Sheves M.. Retinal-carotenoid interactions in a sodium-ion-pumping rhodopsin: Implications on oligomerization and thermal stability. J. Phys. Chem. B. 2023;127(10):2128–2137. doi: 10.1021/acs.jpcb.2c07502. PubMed DOI PMC

Misra R., Eliash T., Sudo Y., Sheves M.. Retinal–salinixanthin interactions in a thermophilic rhodopsin. J. Phys. Chem. B. 2019;123(1):10–20. doi: 10.1021/acs.jpcb.8b06795. PubMed DOI

Kopejtka K., Tomasch J., Kaftan D., Gardiner A. T., Bína D., Gardian Z., Bellas C., Dröge A., Geffers R., Sommaruga R.. et al. A bacterium from a mountain lake harvests light using both proton-pumping xanthorhodopsins and bacteriochlorophyll-based photosystems. Proc. Natl. Acad. Sci. U. S. A. 2022;119(50):e2211018119. doi: 10.1073/pnas.2211018119. PubMed DOI PMC

Chazan A., Das I., Fujiwara T., Murakoshi S., Rozenberg A., Molina-Marquez A., Sano F. K., Tanaka T., Gomez-Villegas P., Larom S.. et al. Phototrophy by antenna-containing rhodopsin pumps in aquatic environments. Nature. 2023;615(7952):535–540. doi: 10.1038/s41586-023-05774-6. PubMed DOI

Fujimoto K. J., Balashov S. P.. Vibronic coupling effect on circular dichroism spectrum: Carotenoid–retinal interaction in xanthorhodopsin. J. Chem. Phys. 2017;146(9):095101. doi: 10.1063/1.4977045. DOI

Heyn M. P., Bauer P. J., Dencher N. A.. A natural cd label to probe the structure of the purple membrane from halobacterium halobium by means of exciton coupling effects. Biochem. Biophys. Res. Commun. 1975;67(3):897–903. doi: 10.1016/0006-291X(75)90761-5. PubMed DOI

Jana S., Jung K.-H., Sheves M.. The chirality origin of retinal-carotenoid complex in gloeobacter rhodopsin: A temperature-dependent excitonic coupling. Sci. Rep. 2020;10(1):13992. doi: 10.1038/s41598-020-70697-5. PubMed DOI PMC

Smolensky E., Sheves M.. Retinal–salinixanthin interactions in xanthorodopsin: A circular dichroism (cd) spectroscopy study with artificial pigments. Biochemistry. 2009;48(34):8179–8188. doi: 10.1021/bi900572b. PubMed DOI

Imasheva E. S., Balashov S. P., Wang J. M., Lanyi J. K.. Removal and reconstitution of the carotenoid antenna of xanthorhodopsin. J. Membr. Biol. 2011;239(1–2):95–104. doi: 10.1007/s00232-010-9322-x. PubMed DOI PMC

Kopejtka K., Tomasch J., Zeng Y., Selyanin V., Dachev M., Piwosz K., Tichy M., Bina D., Gardian Z., Bunk B.. et al. Simultaneous presence of bacteriochlorophyll and xanthorhodopsin genes in a freshwater bacterium. mSystems. 2020;5(6):e01044-20. doi: 10.1128/mSystems.01044-20. PubMed DOI PMC

Simova I., Chrupkova P., Gardiner A. T., Koblizek M., Kloz M., Polivka T.. Femtosecond stimulated raman spectroscopy of linear carotenoids. J. Phys. Chem. Lett. 2024;15(29):7466–7472. doi: 10.1021/acs.jpclett.4c01272. PubMed DOI

Fábryová T., Tůmová L., da Silva D. C., Pereira D. M., Andrade P. B., Valentão P., Hrouzek P., Kopecký J., Cheel J.. Isolation of astaxanthin monoesters from the microalgae haematococcus pluvialis by high performance countercurrent chromatography (HPCCC) combined with high performance liquid chromatography (HPLC) Algal Res. 2020;49:101947. doi: 10.1016/j.algal.2020.101947. DOI

Lutnaes B. F., Oren A., Liaaen-Jensen S.. New C(40)-carotenoid acyl glycoside as principal carotenoid in salinibacter ruber, an extremely halophilic eubacterium. J. Nat. Prod. 2002;65(9):1340–1343. doi: 10.1021/np020125c. PubMed DOI

Lakowicz, J. R. Principles of fluorescence spectroscopy; Springer, 2006.

Humphrey W., Dalke A., Schulten K.. VMD: Visual molecular dynamics. J. Mol. Graph. 1996;14(1):33–38. doi: 10.1016/0263-7855(96)00018-5. PubMed DOI

Neese F.. The ORCA program system. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2012;2(1):73–78. doi: 10.1002/wcms.81. DOI

Neese F., Wennmohs F., Becker U., Riplinger C.. The ORCA quantum chemistry program package. J. Chem. Phys. 2020;152(22):224108. doi: 10.1063/5.0004608. PubMed DOI

Maier J. A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K. E., Simmerling C.. Ff14SB: Improving the 618 accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015;11(8):3696–3713. doi: 10.1021/acs.jctc.5b00255. PubMed DOI PMC

Case D. A., Cheatham T. E. 3rd, Darden T., Gohlke H., Luo R., Merz K. M. Jr., Onufriev A., Simmerling C., Wang B., Woods R. J.. The amber biomolecular simulation programs. J. Comput. Chem. 2005;26(16):1668–1688. doi: 10.1002/jcc.20290. PubMed DOI PMC

Jo S., Kim T., Iyer V. G., Im W.. CHARMM-GUI: A web-based graphical user interface for CHARMM. J. Comput. Chem. 2008;29(11):1859–1865. doi: 10.1002/jcc.20945. PubMed DOI

Metz S., Kästner J., Sokol A. A., Keal T. W., Sherwood P.. Chemshella modular software package for 629 QM/MM simulations. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2014;4(2):101–110. doi: 10.1002/wcms.1163. DOI