Photosynthesis Research

, Volume 136, Issue 2, pp 161–169 | Cite as

Engineering a carotenoid-binding site in Dokdonia sp. PRO95 Na+-translocating rhodopsin by a single amino acid substitution

  • Viktor A. Anashkin
  • Yulia V. Bertsova
  • Adalyat M. Mamedov
  • Mahir D. Mamedov
  • Alexander M. Arutyunyan
  • Alexander A. Baykov
  • Alexander V. Bogachev
Original Article

Abstract

Light-driven H+, Cl and Na+ rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H+-pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na+-pumping rhodopsins (NaRs). We replaced this residue in Dokdonia sp. PRO95 NaR with Gly, which is found in the corresponding position in XRs, and produced a variant rhodopsin in a ketocarotenoid-synthesising Escherichia coli strain. Unlike wild-type NaR, the isolated variant protein contained the tightly bound carotenoids canthaxanthin and echinenone. These carotenoids were visible in the absorption, circular dichroism and fluorescence excitation spectra of the Thr216Gly-substituted NaR, which indicates their function as a light-harvesting antenna. The amino acid substitution and the bound carotenoids did not affect the NaR photocycle. Our findings suggest that the antenna function was recently lost during NaR evolution but can be easily restored by site-directed mutagenesis.

Keywords

Rhodopsin Na+ pump Carotenoid antenna Canthaxanthin Echinenone Xanthorhodopsin 

Abbreviations

CAN

Canthaxanthin

CD

Circular dichroism

DDM

n-Dodecyl β-d-maltoside

ECN

Echinenone

Kr2

Na+-translocating rhodopsin of Krokinobacter eikastus

MD

Molecular dynamics

NaR

Na+-translocating rhodopsin of Dokdonia sp. PRO95

RMSD

Root mean square deviation

SX

Salinixanthin

T216G-NaR

Thr216Gly variant of NaR

XR

Xanthorhodopsin

Notes

Acknowledgements

This work was supported by the Russian Science Foundation Research Project 14-14-00128. We are indebted to Prof. T. Friedrich for providing us the pACCAR25ΔcrtXZcrtO plasmid and for helpful discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Ahl PL, Stern LJ, Mogi T, Khorana HG, Rothschild KJ (1989) Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle. Biochemistry 28:10028–10034CrossRefPubMedGoogle Scholar
  2. Balashov SP, Imasheva ES, Boichenko VA, Antón J, Wang JM, Lanyi JK (2005) Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna. Science 309:2061–2064CrossRefPubMedPubMedCentralGoogle Scholar
  3. Balashov SP, Imasheva ES, Lanyi JK (2006) Induced chirality of the light-harvesting carotenoid salinixanthin and its interaction with the retinal of xanthorhodopsin. Biochemistry 45:10998–11004CrossRefPubMedPubMedCentralGoogle Scholar
  4. Balashov SP, Imasheva ES, Wang JM, Lanyi JK (2008) Excitation energy-transfer and the relative orientation of retinal and carotenoid in xanthorhodopsin. Biophys J 95:2402–2414CrossRefPubMedPubMedCentralGoogle Scholar
  5. Balashov SP, Imasheva ES, Choi AR, Jung KH, Liaaen-Jensen S, Lanyi JK (2010) Reconstitution of Gloeobacter rhodopsin with echinenone: role of the 4-keto group. Biochemistry 49:9792–9799CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bertsova YV, Bogachev AV, Skulachev VP (2015) Proteorhodopsin from Dokdonia sp. PRO95 is a light-driven Na+-pump. Biochemistry (Moscow) 80:449–454CrossRefGoogle Scholar
  7. Bertsova YV, Arutyunyan AM, Bogachev AV (2016) Na+-translocating rhodopsin from Dokdonia sp. PRO95 does not contain carotenoid antenna. Biochemistry (Moscow) 81:414–419CrossRefGoogle Scholar
  8. Bogachev AV, Bertsova YV, Verkhovskaya ML, Mamedov MD, Skulachev VP (2016) Real-time kinetics of electrogenic Na+ transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95. Sci Rep 6:21397CrossRefPubMedPubMedCentralGoogle Scholar
  9. Boichenko VA, Wang JM, Antón J, Lanyi JK, Balashov SP (2006) Functions of carotenoids in xanthorhodopsin and archaerhodopsin from action spectra of photoinhibition of cell respiration. Biochim Biophys Acta 1757:1649–1656CrossRefPubMedPubMedCentralGoogle Scholar
  10. Britton G, Liaaen-Jensen S, Pfander H (2004) Carotenoids handbook, Birkhauser Verlag AG, BaselCrossRefGoogle Scholar
  11. Case DA, Babin V, Berryman JT, Betz RM, Cai Q, Cerutti DS, Cheatham TE III, Darden TA, Duke RE, Gohlke H, Goetz AW, Gusarov S, Homeyer N, Janowski P, Kaus J, Kolossváry I, Kovalenko A, Lee TS, LeGrand S, Luchko T, Luo R, Madej B, Merz KM, Paesani F, Roe DR, Roitberg A, Sagui C, Salomon-Ferrer R, Seabra G, Simmerling CL, Smith W, Swails J, Walker RC, Wang J, Wolf RM, Wu X, Kollman PA (2014) AMBER 14. University of California, San FranciscoGoogle Scholar
  12. Croce R, van Amerongen H (2014) Natural strategies for photosynthetic light harvesting. Nat Chem Biol 10:492–501CrossRefPubMedGoogle Scholar
  13. Fernández-González B, Sandmann G, Vioque A (1997) A new type of asymmetrically acting β-carotene ketolase is required for the synthesis of echinenone in the cyanobacterium Synechocystis sp PCC 6803. J Biol Chem 272:9728–9733CrossRefPubMedGoogle Scholar
  14. Fujimoto KJ, Balashov SP (2017) Vibronic coupling effect on circular dichroism spectrum: carotenoid–retinal interaction in xanthorhodopsin. J Chem Phys 146:095101–095106CrossRefGoogle Scholar
  15. Gushchin I, Shevchenko V, Polovinkin V, Kovalev K, Alekseev A, Round E, Borshchevskiy V, Balandin T, Popov A, Gensch T, Fahlke C, Bamann C, Willbold D, Büldt G, Bamberg E, Gordeliy V (2015) Crystal structure of a light-driven sodium pump. Nat Struct Mol Biol 22:390–395CrossRefPubMedGoogle Scholar
  16. Hsieh LK, Lee TC, Chichester CO, Simpson KL (1974) Biosynthesis of carotenoids in Brevibacterium sp. KY-4313. J Bacteriol 118:385–393PubMedPubMedCentralGoogle Scholar
  17. Imasheva ES, Balashov SP, Wang JM, Smolensky E, Sheves M, Lanyi JK (2008) Chromophore interaction in xanthorhodopsin—retinal dependence of salinixanthin binding. Photochem Photobiol 84:977–984CrossRefPubMedPubMedCentralGoogle Scholar
  18. Imasheva ES, Balashov SP, Choi AR, Jung KH, Lanyi JK (2009) Reconstitution of Gloeobacter violaceus rhodopsin with a light-harvesting carotenoid antenna. Biochemistry 48:10948–10955CrossRefPubMedPubMedCentralGoogle Scholar
  19. Imasheva ES, Balashov SP, Wang JM, Lanyi JK (2011) Removal and reconstitution of the carotenoid antenna of xanthorhodopsin. J Membr Biol 239:95–104CrossRefPubMedGoogle Scholar
  20. Inoue K, Ono H, Abe-Yoshizumi R, Yoshizawa S, Ito H, Kogure K, Kandori H (2013) A light-driven sodium ion pump in marine bacteria. Nat Commun 4:1678CrossRefPubMedGoogle Scholar
  21. Jo S, Kim T, Iyer VG, Im W (2008) CRHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem 29:1859–1865CrossRefPubMedGoogle Scholar
  22. Kato HE, Inoue K, Abe-Yoshizumi R, Kato Y, Ono H, Konno M, Hososhima S, Ishizuka T, Hoque MR, Kunitomo H, Ito J, Yoshizawa S, Yamashita K, Takemoto M, Nishizawa T, Taniguchi R, Kogure K, Maturana AD, Iino Y, Yawo H, Ishitani R, Kandori H, Nureki O (2015) Structural basis for Na+ transport mechanism by a light-driven Na+ pump. Nature 521:48–53CrossRefPubMedGoogle Scholar
  23. Luecke H, Schobert B, Stagno J, Imasheva ES, Wang JM, Balashov SP, Lanyi JK (2008) Crystallographic structure of xanthorhodopsin the light-driven proton pump with a dual chromophore. Proc Natl Acad Sci USA 105:16561–16565CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lutnaes BF, Oren A, Liaaen-Jensen S (2002) New C40-carotenoid acyl glycoside as principal carotenoid in Salinibacter ruber, an extremely halophilic eubacterium. J Nat Prod 65:1340–1343CrossRefPubMedGoogle Scholar
  25. Mamedov MD, Mamedov AM, Bertsova YV, Bogachev AV (2016) A single mutation converts bacterial Na+-transporting rhodopsin into an H+ transporter. FEBS Lett 590:2827–2835CrossRefPubMedGoogle Scholar
  26. Marti T, Otto H, Mogi T, Rösselet SJ, Heyn MP, Khorana HG (1991) Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation. J Biol Chem 266:6919–6927PubMedGoogle Scholar
  27. Moldenhauer M, Sluchanko NN, Buhrke D, Zlenko DV, Tavraz NN, Schmitt FJ, Hildebrandt P, Maksimov EG, Friedrich T (2017) Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism. Photosynth Res 133:327–341CrossRefPubMedGoogle Scholar
  28. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefPubMedGoogle Scholar
  29. Smolensky Koganov E, Brumfeld V, Friedman N, Sheves M (2015) Origin of circular dichroism of xanthorhodopsin. A study with artificial pigments. J Phys Chem B 119:456–464CrossRefPubMedGoogle Scholar
  30. Tinoco I (1963) The exciton contribution to the optical rotation of polymers. Radiat Res 20:133–139CrossRefGoogle Scholar
  31. Vollmers J, Voget S, Dietrich S, Gollnow K, Smits M, Meyer K, Brinkhoff T, Simon M, Daniel R (2013) Poles apart: arctic and Antarctic Octadecabacter strains share high genome plasticity and a new type of xanthorhodopsin. PLoS ONE 8:e63422CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia

Personalised recommendations