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The elusive relationship between structure and colour emission in beetle luciferases

Abstract

In beetles, luciferase enzymes catalyse the conversion of chemical energy into light through bioluminescence. The principles of this process have become a fundamental biotechnological tool that revolutionized biological research. Different beetle species can emit different colours of light, despite using the same substrate and highly homologous luciferases. The chemical reasons for these different colours are hotly debated yet remain unresolved. This Review summarizes the structural, biochemical and spectrochemical data on beetle bioluminescence reported over the past three decades. We identify the factors that govern what colour is emitted by wild-type and mutant luciferases. This topic is controversial, but, in general, we note that green emission requires cationic residues in a specific position near the benzothiazole fragment of the emitting molecule, oxyluciferin. The commonly emitted green–yellow light can be readily changed to red by introducing a variety of individual and multiple mutations. However, complete switching of the emitted light from red to green has not been accomplished and the synergistic effects of combined mutations remain unexplored. The minor colour shifts produced by most known mutations could be important in establishing a ‘mutational catalogue’ to fine-tune emission of beetle luciferases, thereby expanding the scope of their applications.

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Fig. 1: Bioluminescent beetles and the chemical basis for their emission across the visible spectrum.
Fig. 2: Different luciferases operate on the same substrates but emit different wavelengths, as explained in six mechanistic proposals.
Fig. 3: Structures of luciferases from different beetle species.
Fig. 4: Structures of different luciferases bound to reaction products or model reaction intermediates reveal enzymatic changes during bioluminescence.
Fig. 5: Chemical structures of relevant luciferases inhibitors that have been structurally resolved in complex with FLuc.
Fig. 6: Colour-shifting mutations in the structure of a green-emitting luciferase.
Fig. 7: Mutations can affect the colour emitted by naturally green-emitting and naturally red-emitting luciferases.
Fig. 8: Mutations can affect the colour emitted by two naturally green-emitting luciferases.

References

  1. 1.

    Newton, H. E. A History Lumin. Earliest 1900. Memoirs of the American Philosophical Society Vol. 44 (J. H. Furst Company, 1958).

  2. 2.

    Liu, Y.-J., De Vico, L. & Lindh, R. Ab initio investigation on the chemical origin of the firefly bioluminescence. J. Photochem. Photobiol. A 194, 261–267 (2008).

    CAS  Google Scholar 

  3. 3.

    Liu, F., Liu, Y., De Vico, L. & Lindh, R. Theoretical study of the chemiluminescent decomposition of dioxetanone. J. Am. Chem. Soc. 131, 6181–6188 (2009).

    CAS  PubMed  Google Scholar 

  4. 4.

    Navizet, I. et al. Color-tuning mechanism of firefly investigated by multi-configurational perturbation method. J. Am. Chem. Soc. 132, 706–712 (2010).

    CAS  PubMed  Google Scholar 

  5. 5.

    Chen, S.-F. et al. Systematic theoretical investigation on the light emitter of firefly. J. Chem. Theory Comput. 7, 798–803 (2011).

    CAS  PubMed  Google Scholar 

  6. 6.

    Yue, L., Roca-Sanjuán, D., Lindh, R., Ferré, N. & Liu, Y.-J. Can the closed-shell DFT methods describe the thermolysis of 1,2-dioxetanone? J. Chem. Theory Comput. 8, 4359–4363 (2012).

    CAS  PubMed  Google Scholar 

  7. 7.

    Navizet, I. et al. Are the bio- and chemiluminescence states of the firefly oxyluciferin the same as the fluorescence state? Photochem. Photobiol. 89, 319–325 (2013).

    CAS  PubMed  Google Scholar 

  8. 8.

    Augusto, F. A. et al. Mechanism of activated chemiluminescence of cyclic peroxides: 1,2-dioxetanes and 1,2-dioxetanones. Phys. Chem. Chem. Phys. 19, 3955–3962 (2017).

    CAS  PubMed  Google Scholar 

  9. 9.

    Francés-Monerris, A., Galván, I. F., Lindh, R. & Roca-Sanjuán, D. Triplet versus singlet chemiexcitation mechanism in dioxetanone: a CASSCF/CASPT2 study. Theor. Chem. Acc. 136, 70 (2017).

    Google Scholar 

  10. 10.

    Berraud-Pache, R., Lindh, R. & Navizet, I. QM/MM study of the formation of the dioxetanone ring in fireflies through a superoxide ion. J. Phys. Chem. B 122, 5173–5182 (2018).

    CAS  PubMed  Google Scholar 

  11. 11.

    Vacher, M. et al. Chemi- and bioluminescence of cyclic peroxides. Chem. Rev. 118, 6927–6974 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    McElroy, W. D. The energy source for bioluminescence in an isolated system. Proc. Natl Acad. Sci. USA 33, 342–345 (1947).

    CAS  PubMed  Google Scholar 

  13. 13.

    Strehler, B. L. & Totter, J. R. in Methods of Biochemical Analysis (ed Glick, D.) 341–356 (Wiley, 1954).

  14. 14.

    Lundin, A. & Thore, A. Analytical information obtainable by evaluation of the time course of firefly bioluminescence in the assay of ATP. Anal. Biochem. 66, 47–63 (1975).

    CAS  PubMed  Google Scholar 

  15. 15.

    Hysert, D. W., Kovecses, F. & Morrison, N. M. A firefly bioluminescence ATP assay method for rapid detection and enumeration of brewery microorganisms. J. Am. Soc. Brew. Chem. 34, 145–150 (1976).

    CAS  Google Scholar 

  16. 16.

    Beigi, R., Kobatake, E., Aizawa, M. & Dubyak, G. R. Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am. J. Physiol. 276, C267–C278 (1999).

    CAS  PubMed  Google Scholar 

  17. 17.

    Mezzanotte, L., van ‘t Root, M., Karatas, H., Goun, E. A. & Löwik, C. W. G. M. In vivo molecular bioluminescence imaging: new tools and applications. Trends Biotechnol. 35, 640–652 (2017).

    CAS  PubMed  Google Scholar 

  18. 18.

    Maric, T. et al. Bioluminescent-based imaging and quantification of glucose uptake in vivo. Nat. Methods 16, 526–532 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Momota, H. & Holland, E. C. Bioluminescence technology for imaging cell proliferation. Curr. Opin. Biotechnol. 16, 681–686 (2005).

    CAS  PubMed  Google Scholar 

  20. 20.

    Contag, C. H. & Bachmann, M. H. Advances in in vivo bioluminescence imaging of gene expression. Annu. Rev. Biomed. Eng. 4, 235–260 (2002).

    CAS  PubMed  Google Scholar 

  21. 21.

    Steinberg, S. M., Poziomek, E. J., Engelmann, W. H. & Rogers, K. R. A review of environmental applications of bioluminescence measurements. Chemosphere 30, 2155–2197 (1995).

    CAS  Google Scholar 

  22. 22.

    Fernández-Piñas, F., Rodea-Palomares, I., Leganés, F., González-Pleiter, M. & Muñoz-Martín, M. A. Evaluation of the ecotoxicity of pollutants with bioluminescent microorganisms. Adv. Biochem. Eng. Biotechnol. 145, 65–135 (2014).

    PubMed  Google Scholar 

  23. 23.

    Alloush, H. M., Lewis, R. J. & Salisbury, V. C. Bacterial bioluminescent biosensors: applications in food and environmental monitoring. Anal. Lett. 39, 1517–1526 (2006).

    CAS  Google Scholar 

  24. 24.

    Santos-Merino, M., Singh, A. K. & Ducat, D. C. New applications of synthetic biology tools for cyanobacterial metabolic engineering. Front. Bioeng. Biotechnol. 7, 1–24 (2019).

    Google Scholar 

  25. 25.

    Khakhar, A. et al. Building customizable auto-luminescent luciferase-based reporters in plants. eLife 9, e52786 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Fisher, A. J., Rayment, I., Raushel, F. M. & Baldwin, T. O. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 Å resolution. Biochemistry 34, 6581–6586 (1995).

    CAS  PubMed  Google Scholar 

  27. 27.

    Conti, E., Franks, N. P. & Brick, P. Crystal structure of firefly luciferase throws light on a super-family of adenylate-forming enzymes. Structure 4, 287–298 (1996).

    CAS  PubMed  Google Scholar 

  28. 28.

    Nakatsu, T. et al. Structural basis for the spectral difference in luciferase bioluminescence. Nature 440, 372–376 (2006).

    CAS  PubMed  Google Scholar 

  29. 29.

    Naumov, P., Ozawa, Y., Ohkubo, K. & Fukuzumi, S. Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence. J. Am. Chem. Soc. 131, 11590–11605 (2009).

    CAS  PubMed  Google Scholar 

  30. 30.

    Maltsev, O. V., Nath, N. K., Naumov, P. & Hintermann, L. Why is firefly oxyluciferin a notoriously labile substance? Angew. Chem. Int. Ed. 53, 847–850 (2014).

    CAS  Google Scholar 

  31. 31.

    Carrasco-López, C. et al. Beetle luciferases with naturally red- and blue-shifted emission. Life Sci. Alliance 1, 1–10 (2018).

    Google Scholar 

  32. 32.

    DeLuca, M. & McElroy, W. D. Kinetics of the firefly luciferase catalyzed reactions. Biochemistry 13, 921–925 (1974).

    CAS  PubMed  Google Scholar 

  33. 33.

    Ugarova, N. N., Brovko, Y. L. & Berezin, I. V. Immobilized firefly luciferase and its use in analysis. Anal. Lett. 13, 881–892 (1980).

    CAS  Google Scholar 

  34. 34.

    Said Alipour, B. et al. Molecular cloning, sequence analysis, and expression of a cDNA encoding the luciferase from the glow-worm, Lampyris turkestanicus. Biochem. Biophys. Res. Commun. 325, 215–222 (2004).

    CAS  Google Scholar 

  35. 35.

    Ugarova, N. N., Maloshenok, L. G., Uporov, I. V. & Koksharov, M. I. Bioluminescence spectra of native and mutant firefly luciferases as a function of pH. Biochemistry 70, 1262–1267 (2005).

    CAS  PubMed  Google Scholar 

  36. 36.

    Viviani, V. R., Arnoldi, F. G. C., Brochetto-Braga, M. & Ohmiya, Y. Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 139, 151–156 (2004).

    CAS  PubMed  Google Scholar 

  37. 37.

    Viviani, V. R., Oehlmeyer, T. L., Arnoldi, F. G. C. & Brochetto-Braga, M. R. A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases. Photochem. Photobiol. 81, 843–848 (2005).

    CAS  PubMed  Google Scholar 

  38. 38.

    Viviani, V. R., Amaral, D., Prado, R. & Arnoldi, F. G. C. A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties. Photochem. Photobiol. Sci. 10, 1879–1886 (2011).

    CAS  PubMed  Google Scholar 

  39. 39.

    Amaral, D. T., Oliveira, G., Silva, J. R. & Viviani, V. R. A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations. Photochem. Photobiol. Sci. 15, 1148–1154 (2016).

    CAS  PubMed  Google Scholar 

  40. 40.

    Branchini, B. R. et al. Cloning of the orange light-producing luciferase from Photinus scintillans — a new proposal on how bioluminescence color is determined. Photochem. Photobiol. 93, 479–485 (2017).

    CAS  PubMed  Google Scholar 

  41. 41.

    Ugarova, N. N., Brovko, L. Y. & Beliaieva, E. I. Immobilization of luciferase from the firefly Luciola mingrelica: catalytic properties and thermostability of the enzyme immobilized on cellulose films. Enzyme Microb. Technol. 5, 60–64 (1983).

    CAS  Google Scholar 

  42. 42.

    Brovko, L., Beliaeva, E. I. & Ugarova, N. N. Subunit interactions in luciferase from the firefly Luciola mingrelica. Their role in the manifestation of enzyme activity and during thermoinactivation. Biochemistry 47, 760–766 (1982).

    CAS  Google Scholar 

  43. 43.

    Filippova, N. Y., Dukhovich, A. F. & Ugarova, N. N. New approaches to the preparation and application of firefly luciferase. J. Biolumin. Chemilumin. 4, 419–422 (1989).

    CAS  PubMed  Google Scholar 

  44. 44.

    Tatsumi, H., Masuda, T., Kajiyama, N. & Nakano, E. Luciferase cDNA from Japanese firefly, Luciola cruciata: cloning, structure and expression in Escherichia coli. J. Biolumin. Chemilumin. 3, 75–78 (1989).

    CAS  PubMed  Google Scholar 

  45. 45.

    Devine, J. H., Kutuzova, G. D., Green, V. A., Ugarova, N. N. & Baldwin, T. O. Luciferase from the East European firefly Luciola mingrelica: cloning and nucleotide sequence of the cDNA, overexpression in Escherichia coli and purification of the enzyme. Biochim. Biophys. Acta Gene Struct. Expr. 1173, 121–132 (1993).

    CAS  Google Scholar 

  46. 46.

    Viviani, V. R. & Bechara, E. J. H. Bioluminescence of Brazilian fireflies (Coleoptera: Lampyridae): spectral distribution and pH effect on luciferase-elicited colors. Comparison with elaterid and phengodid luciferases. Photochem. Photobiol. 62, 490–495 (1995).

    CAS  Google Scholar 

  47. 47.

    Viviani, V. R., Bechara, E. J. H. & Ohmiya, Y. Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures. Biochemistry 38, 8271–8279 (1999).

    CAS  PubMed  Google Scholar 

  48. 48.

    Viviani, V. R. et al. Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase. Photochem. Photobiol. 70, 254–260 (1999).

    CAS  PubMed  Google Scholar 

  49. 49.

    Branchini, B. R. et al. An alternative mechanism of bioluminescence color determination in firefly luciferase. Biochemistry 43, 7255–7262 (2004).

    CAS  PubMed  Google Scholar 

  50. 50.

    Viviani, V. R. et al. Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases. J. Biochem. 140, 467–474 (2006).

    CAS  PubMed  Google Scholar 

  51. 51.

    Tafreshi, N. K. et al. The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase. J. Biol. Chem. 282, 8641–8647 (2007).

    CAS  PubMed  Google Scholar 

  52. 52.

    Viviani, V. R., Silva Neto, A. J., Arnoldi, F. G. C., Barbosa, J. A. R. G. & Ohmiya, Y. The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: A solvent gate for the active site of pH-sensitive luciferases. Photochem. Photobiol. 84, 138–144 (2008).

    CAS  PubMed  Google Scholar 

  53. 53.

    Koksharov, M. I. & Ugarova, N. N. Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity. Biochemistry 73, 862–869 (2008).

    CAS  PubMed  Google Scholar 

  54. 54.

    Said Alipour, B., Hosseinkhani, S., Ardestani, S. K. & Moradi, A. The effective role of positive charge saturation in bioluminescence color and thermostability of firefly luciferase. Photochem. Photobiol. Sci. 8, 847–855 (2009).

    CAS  PubMed  Google Scholar 

  55. 55.

    Hirano, T. et al. Spectroscopic studies of the color modulation mechanism of firefly (beetle) bioluminescence with amino-analogs of luciferin and oxyluciferin. Photochem. Photobiol. Sci. 11, 1281–1284 (2012).

    CAS  PubMed  Google Scholar 

  56. 56.

    Viviani, V. R. et al. Glu311 and Arg337 stabilize a closed active-site conformation and provide a critical catalytic base and countercation for green bioluminescence in beetle luciferases. Biochemistry 55, 4764–4776 (2016).

    CAS  PubMed  Google Scholar 

  57. 57.

    Bevilaqua, V. R. et al. Phrixotrix luciferase and 6′-aminoluciferins reveal a larger luciferin phenolate binding site and provide novel far-red combinations for bioimaging purposes. Sci. Rep. 9, 8998 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Modestova, Y. & Ugarova, N. N. Color-shifting mutations in the C-domain of L. mingrelica firefly luciferase provide new information about the domain alternation mechanism. Biochim. Biophys. Acta Proteins Proteom. 1864, 1818–1826 (2016).

    CAS  Google Scholar 

  59. 59.

    Branchini, B. R. et al. Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color. Biochemistry 38, 13223–13230 (1999).

    CAS  PubMed  Google Scholar 

  60. 60.

    Viviani, V. R. & Ohmiya, Y. Bioluminescence color determinants of Phrixothrix railroad-worm luciferases: chimeric luciferases, site-directed mutagenesis of Arg 215 and guanidine effect. Photochem. Photobiol. 72, 267–271 (2000).

    CAS  PubMed  Google Scholar 

  61. 61.

    Viviani, V., Uchida, A., Suenaga, N., Ryufuku, M. & Ohmiya, Y. Thr226 is a key residue for bioluminescence spectra determination in beetle luciferases. Biochem. Biophys. Res. Commun. 280, 1286–1291 (2001).

    CAS  PubMed  Google Scholar 

  62. 62.

    Viviani, V. R., Uchida, A., Viviani, W. & Ohmiya, Y. The influence of Ala243 (Gly247), Arg215 and Thr226 (Asn230) on the bioluminescence spectra and pH-sensitivity of railroad worm, click beetle and firefly luciferases. Photochem. Photobiol. 76, 538–544 (2002).

    CAS  PubMed  Google Scholar 

  63. 63.

    Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H. & Fleet, S. E. A mutagenesis study of the putative luciferin binding site residues of firefly luciferase. Biochemistry 42, 10429–10436 (2003).

    CAS  PubMed  Google Scholar 

  64. 64.

    Solntsev, K. M., Laptenok, S. P. & Naumov, P. Photoinduced dynamics of oxyluciferin analogues: Unusual enol “super” photoacidity and evidence for keto–enol isomerization. J. Am. Chem. Soc. 134, 16452–16455 (2012).

    CAS  PubMed  Google Scholar 

  65. 65.

    Naumov, P. & Kochunnoonny, M. Spectral–structural effects of the keto–enol–enolate and phenol–phenolate equilibria of oxyluciferin. J. Am. Chem. Soc. 132, 11566–11579 (2010).

    CAS  PubMed  Google Scholar 

  66. 66.

    Ghose, A. et al. Emission properties of oxyluciferin and its derivatives in water: revealing the nature of the emissive species in firefly bioluminescence. J. Phys. Chem. B 119, 2638–2649 (2015).

    CAS  PubMed  Google Scholar 

  67. 67.

    Snellenburg, J. J., Laptenok, S. P., Desa, R. J., Naumov, P. & Solntsev, K. M. Excited-state dynamics of oxyluciferin in firefly luciferase. J. Am. Chem. Soc. 138, 16252–16258 (2016).

    CAS  PubMed  Google Scholar 

  68. 68.

    Saleh, N. et al. Bioinspired molecular lantern: tuning the firefly oxyluciferin emission with host–guest chemistry. J. Phys. Chem. B 120, 7671–7680 (2016).

    CAS  PubMed  Google Scholar 

  69. 69.

    Støchkel, K. et al. On the influence of water on the electronic structure of firefly oxyluciferin anions from absorption spectroscopy of bare and monohydrated ions in vacuo. J. Am. Chem. Soc. 135, 6485–6493 (2013).

    PubMed  Google Scholar 

  70. 70.

    Rebarz, M. et al. Deciphering the protonation and tautomeric equilibria of firefly oxyluciferin by molecular engineering and multivariate curve resolution. Chem. Sci. 4, 3803–3809 (2013).

    CAS  Google Scholar 

  71. 71.

    Ando, Y. et al. Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission. Nat. Photonics 2, 44–47 (2008).

    CAS  Google Scholar 

  72. 72.

    Ando, Y. & Akiyama, H. pH-dependent fluorescence spectra, lifetimes, and quantum yields of firefly-luciferin aqueous solutions studied by selective-excitation fluorescence spectroscopy. Jpn. J. Appl. Phys. 49, 117002 (2010).

    Google Scholar 

  73. 73.

    Hiyama, M., Akiyama, H., Yamada, K. & Koga, N. Theoretical study of firefly luciferin pKa values — relative absorption intensity in aqueous solutions. Photochem. Photobiol. 89, 571–578 (2013).

    CAS  PubMed  Google Scholar 

  74. 74.

    Wang, Y., Akiyama, H., Terakado, K. & Nakatsu, T. Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence. Sci. Rep. 3, 2490 (2013).

    PubMed  PubMed Central  Google Scholar 

  75. 75.

    Wang, Y., Hayamizu, Y. & Akiyama, H. Spectroscopic study of firefly oxyluciferin in an enzymatic environment on the basis of stability monitoring. J. Phys. Chem. B 118, 2070–2076 (2014).

    CAS  PubMed  Google Scholar 

  76. 76.

    Mochizuki, T., Wang, Y., Hiyama, M. & Akiyama, H. Robust red-emission spectra and yields in firefly bioluminescence against temperature changes. Appl. Phys. Lett. 104, 213704 (2014).

    Google Scholar 

  77. 77.

    Hirano, T. et al. Spectroscopic studies of the light-color modulation mechanism of firefly (beetle) bioluminescence. J. Am. Chem. Soc. 131, 2385–2396 (2009).

    CAS  PubMed  Google Scholar 

  78. 78.

    Song, C. I. & Rhee, Y. M. Dynamics on the electronically excited state surface of the bioluminescent firefly luciferase–oxyluciferin system. J. Am. Chem. Soc. 133, 12040–12049 (2011).

    CAS  PubMed  Google Scholar 

  79. 79.

    Kim, H. W. & Rhee, Y. M. On the pH dependent behavior of the firefly bioluminescence: protein dynamics and water content in the active pocket. J. Phys. Chem. B 117, 7260–7269 (2013).

    CAS  PubMed  Google Scholar 

  80. 80.

    da Silva, L. P. & Esteves da Silva, J. C. G. Computational studies of the luciferase light-emitting product: oxyluciferin. J. Chem. Theory Comput. 7, 809–817 (2011).

    PubMed  Google Scholar 

  81. 81.

    Pinto da Silva, L., Vieira, J. & Esteves da Silva, J. C. G. Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase. Chem. Phys. Lett. 543, 137–141 (2012).

    CAS  Google Scholar 

  82. 82.

    da Silva, L., Simkovitch, R., Huppert, D. & da Silva, J. C. G. Theoretical photodynamic study of the photoprotolytic cycle of firefly oxyluciferin. ChemPhysChem 14, 2711–2716 (2013).

    Google Scholar 

  83. 83.

    Yue, L., Lan, Z. & Liu, Y.-J. The theoretical estimation of the bioluminescent efficiency of the firefly via a nonadiabatic molecular dynamics simulation. J. Phys. Chem. Lett. 6, 540–548 (2015).

    CAS  PubMed  Google Scholar 

  84. 84.

    Cheng, Y.-Y. & Liu, Y.-J. Vibrationally resolved absorption and fluorescence spectra of firefly luciferin: a theoretical simulation in the gas phase and in solution. Photochem. Photobiol. 92, 552–560 (2016).

    CAS  PubMed  Google Scholar 

  85. 85.

    White, E. H., Rapaport, E., Hopkins, T. A. & Seliger, H. H. Chemi- and bioluminescence of firefly luciferin. J. Am. Chem. Soc. 91, 2178–2180 (1969).

    CAS  PubMed  Google Scholar 

  86. 86.

    White, E. H., Rapaport, E., Seliger, H. H. & Hopkins, T. A. The chemi- and bioluminescence of firefly luciferin: an efficient chemical production of electronically excited states. Bioorg. Chem. 1, 92–122 (1971).

    CAS  Google Scholar 

  87. 87.

    McCapra, F., Gilfoyle, D. J., Young, D. W., Church, N. J. & Spencer, P. in Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects Vol. 387 (eds Campbell, A., Kricka, L. J. & Stanley, P. E.) (Wiley, 1994).

  88. 88.

    Tianxiao, Y. & Goddard, J. D. Predictions of the geometries and fluorescence emission energies of oxyluciferins. J. Phys. Chem. A 111, 4489–4497 (2007).

    Google Scholar 

  89. 89.

    Nakatani, N., Hasegawa, J.-y. & Nakatsuji, H. Red light in chemiluminescence and yellow-green light in bioluminescence: color-tuning mechanism of firefly, Photinus pyralis, studied by the symmetry-adapted cluster–configuration interaction method. J. Am. Chem. Soc. 129, 8756–8765 (2007).

    CAS  PubMed  Google Scholar 

  90. 90.

    Ugarova, N. N. & Brovko, L. Y. Protein structure and bioluminescent spectra for firefly bioluminescence. Luminescence 17, 321–330 (2002).

    CAS  PubMed  Google Scholar 

  91. 91.

    Gandelman, O. A., Brovko, L. Y., Ugarova, N. N., Chikishev, A. Y. & Shkurimov, A. P. Oxyluciferin fluorescence is a model of native bioluminescence in the firefly luciferin–luciferase system. J. Photochem. Photobiol. B 19, 187–191 (1993).

    CAS  Google Scholar 

  92. 92.

    Gandelman, O. A., Brovko, L. Y., Chikishev, A. Y., Shkurinov, A. P. & Ugarova, N. N. Investigation of the interaction between firefly luciferase and oxyluciferin or its analogues by steady state and subnanosecond time-resolved fluorescence. J. Photochem. Photobiol. B 22, 203–209 (1994).

    CAS  Google Scholar 

  93. 93.

    Sandalova, T. P. & Ugarova, N. N. Model of the active site of firefly luciferase. Biochemistry 64, 962–967 (1999).

    CAS  PubMed  Google Scholar 

  94. 94.

    Ugarova, N. N. & Brovko, L. Y. Relationship between the structure of the protein globule and bioluminescence spectra of firefly luciferase. Russ. Chem. Bull. 50, 1752–1761 (2001).

    CAS  Google Scholar 

  95. 95.

    Branchini, B. R., Murtiashaw, M. H., Magyar, R. A. & Anderson, S. M. The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase. Biochemistry 39, 5433–5440 (2000).

    CAS  PubMed  Google Scholar 

  96. 96.

    Sundlov, J. A., Fontaine, D. M., Southworth, T. L., Branchini, B. R. & Gulick, A. M. Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism. Biochemistry 51, 6493–6495 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. 97.

    Branchini, B. R. et al. Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism. J. Am. Chem. Soc. 133, 11088–11091 (2011).

    CAS  PubMed  Google Scholar 

  98. 98.

    Zako, T. et al. Luminescent and substrate binding activities of firefly luciferase N-terminal domain. Biochim. Biophys. Acta Proteins Proteom. 1649, 183–189 (2003).

    CAS  Google Scholar 

  99. 99.

    Ayabe, K., Zako, T. & Ueda, H. The role of firefly luciferase C-terminal domain in efficient coupling of adenylation and oxidative steps. FEBS Lett. 579, 4389–4394 (2005).

    CAS  PubMed  Google Scholar 

  100. 100.

    Koksharov, M. I. & Ugarova, N. N. Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence. Photochem. Photobiol. Sci. 12, 2016–2027 (2013).

    CAS  PubMed  Google Scholar 

  101. 101.

    Lee, J. in Bioluminescence, the Nature of the Light 107–121 (Univ. Georgia, 2015).

  102. 102.

    Seliger, H. H. & McElroy, W. D. The colors of firefly bioluminescence: enzyme configuration and species specificity. Proc. Natl Acad. Sci. USA 52, 75–81 (1964).

    CAS  PubMed  Google Scholar 

  103. 103.

    Wood, K. V., Lam, Y. A. & McElroy, W. D. Introduction to beetle luciferases and their applications. J. Biolumin. Chemilumin. 4, 289–301 (1989).

    CAS  PubMed  Google Scholar 

  104. 104.

    Ueda, I., Shinoda, F. & Kamaya, H. Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase. Biophys. J. 66, 2107–2110 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. 105.

    Zhao, H. et al. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J. Biomed. Opt. 10, 041210 (2005).

    Google Scholar 

  106. 106.

    Kajiyama, N. & Nakano, E. Isolation and characterization of mutants of firefly luciferase which produce different colors of light. Protein Eng. Des. Sel. 4, 691–693 (1991).

    CAS  Google Scholar 

  107. 107.

    Cruz, P. G. et al. Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors. Chem. Biol. 18, 1442–1452 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. 108.

    Auld, D. S. et al. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124. Proc. Natl Acad. Sci. USA 107, 4878–4883 (2010).

    CAS  PubMed  Google Scholar 

  109. 109.

    Franks, N. P., Jenkins, A., Conti, E., Lieb, W. R. & Brick, P. Structural basis for the inhibition of firefly luciferase by a general anesthetic. Biophys. J. 75, 2205–2211 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. 110.

    Zhang, H. et al. Inhibiting firefly bioluminescence by chalcones. Anal. Chem. 89, 6099–6105 (2017).

    CAS  PubMed  Google Scholar 

  111. 111.

    Thorne, N., Inglese, J. & Auld, D. S. Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem. Biol. 17, 646–657 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. 112.

    Thorne, N. et al. Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem. Biol. 19, 1060–1072 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113.

    Auld, D. S. & Inglese, J. in Assay Guidance Manual (eds Markossian, S. et al) (Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004).

  114. 114.

    Mamaev, S. V., Laikhter, A. L., Arslan, T. & Hecht, S. M. Firefly luciferase: alteration of the color of emitted light resulting from substitutions at position 286. J. Am. Chem. Soc. 118, 7243–7244 (1996).

    CAS  Google Scholar 

  115. 115.

    Maghami, P. et al. Relationship between stability and bioluminescence color of firefly luciferase. Photochem. Photobiol. Sci. 9, 376–383 (2010).

    CAS  PubMed  Google Scholar 

  116. 116.

    Kheirabadi, M. et al. Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift. Biochim. Biophys. Acta 1834, 2729–2735 (2013).

    CAS  PubMed  Google Scholar 

  117. 117.

    Nishiguchi, T. et al. Development of red-shifted mutants derived from luciferase of Brazilian click beetle Pyrearinus termitilluminans. J. Biomed. Opt. 20, 101205 (2015).

    PubMed  Google Scholar 

  118. 118.

    Li, X., Nakajima, Y., Niwa, K., Viviani, V. R. & Ohmiya, Y. Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging. Protein Sci. 19, 26–33 (2010).

    PubMed  Google Scholar 

  119. 119.

    Koksharov, M. I. & Ugarova, N. N. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein Eng. Des. Sel. 24, 835–844 (2011).

    CAS  PubMed  Google Scholar 

  120. 120.

    Koksharov, M. I. & Ugarova, N. N. Approaches to engineer stability of beetle luciferases. Comput. Struct. Biotechnol. J. 2, e201204004 (2012).

    Google Scholar 

  121. 121.

    Koksharov, M. I. & Ugarova, N. N. Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelica firefly luciferase. Photochem. Photobiol. Sci. 10, 931–938 (2011).

    CAS  PubMed  Google Scholar 

  122. 122.

    Oliveira, G. & Viviani, V. R. Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: relationship with kinetics and bioluminescence colours. Luminescence 33, 282–288 (2018).

    CAS  PubMed  Google Scholar 

  123. 123.

    Sarrion-Perdigones, A. et al. Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying. Nat. Commun. 10, 5710 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. 124.

    Ohmiya, Y. Simultaneous multicolor luciferase reporter assays for monitoring of multiple genes expressions. Comb. Chem. High Throughput Screen. 18, 937–945 (2015).

    CAS  PubMed  Google Scholar 

  125. 125.

    Carrasco-López, C., García-Echauri, S. A., Kichuk, T. & Avalos, J. L. Optogenetics and biosensors set the stage for metabolic cybergenetics. Curr. Opin. Biotechnol. 65, 296–309 (2020).

    PubMed  Google Scholar 

  126. 126.

    Cheng, Y.-Y. & Liu, Y.-J. What exactly is the light emitter of a firefly? J. Chem. Theory Comput. 11, 5360–5370 (2015).

    CAS  PubMed  Google Scholar 

  127. 127.

    Cheng, Y.-Y. & Liu, Y.-J. Theoretical development of near-infrared bioluminescent systems. Chem. Eur. J. 24, 9340–9352 (2018).

    CAS  PubMed  Google Scholar 

  128. 128.

    Wu, W. et al. cybLuc: an effective aminoluciferin derivative for deep bioluminescence imaging. Anal. Chem. 89, 4808–4816 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  129. 129.

    Branchini, B. R. et al. Mutagenesis and structural studies reveal the basis for the activity and stability properties that distinguish the Photinus luciferases scintillans and pyralis. Biochemistry 58, 4293–4303 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. 130.

    Stowe, C. L. et al. Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin. eLife 8, e45801 (2019).

    PubMed  PubMed Central  Google Scholar 

  131. 131.

    Pelentir, G. F., Bevilaqua, V. R. & Viviani, V. R. A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays. Photochem. Photobiol. Sci. 18, 2061–2070 (2019).

    CAS  PubMed  Google Scholar 

  132. 132.

    Arnoldi, F. G. C., da Silva Neto, A. J. & Viviani, V. R. Molecular insights on the evolution of the lateral and head lantern luciferases and bioluminescence colors in Mastinocerini railroad-worms (Coleoptera: Phengodidae). Photochem. Photobiol. Sci. 9, 87–92 (2010).

    CAS  PubMed  Google Scholar 

  133. 133.

    Branchini, B. R. et al. Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal. Biochem. 361, 253–262 (2007).

    CAS  PubMed  Google Scholar 

  134. 134.

    Branchini, B. R. et al. Red-emitting luciferases for bioluminescence reporter and imaging applications. Anal. Biochem. 396, 290–297 (2010).

    CAS  PubMed  Google Scholar 

  135. 135.

    Branchini, B. R. et al. Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain. Biochemistry 44, 1385–1393 (2005).

    CAS  PubMed  Google Scholar 

  136. 136.

    Nazari, M., Hosseinkhani, S. & Hassani, L. Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective. Photochem. Photobiol. Sci. 12, 298–308 (2013).

    CAS  PubMed  Google Scholar 

  137. 137.

    White, P. J., Squirrell, D. J., Arnaud, P., Lowe, C. R. & Murray, J. A. H. Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354. Biochem. J. 319, 343–350 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. 138.

    Caysa, H. et al. A redshifted codon-optimized firefly luciferase is a sensitive reporter for bioluminescence imaging. Photochem. Photobiol. Sci. 8, 52–56 (2009).

    CAS  PubMed  Google Scholar 

  139. 139.

    Shapiro, E., Lu, C. & Baneyx, F. A set of multicolored Photinus pyralis luciferase mutants for in vivo bioluminescence applications. Protein Eng. Des. Sel. 18, 581–587 (2005).

    CAS  PubMed  Google Scholar 

  140. 140.

    Branchini, B. R., Southworth, T. L., Khattak, N. F., Murtiashaw, M. H. & Fleet, S. E. in Genetically Engineered and Optical Probes for Biomedical Applications II Vol. 5329 (eds Savitsky, A. P., Brovko, L. Y., Bornhop, D. J., Raghavachari, R. & Achilefu, S. I.) (International Society for Optics and Photonics, 2004).

  141. 141.

    Branchini, B. R., Southworth, T. L., Khattak, N. F., Michelini, E. & Roda, A. Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal. Biochem. 345, 140–148 (2005).

    CAS  PubMed  Google Scholar 

  142. 142.

    Branchini, B. R., Magyar, R. A., Murtiashaw, M. H. & Portier, N. C. The role of active site residue arginine 218 in firefly luciferase bioluminescence. Biochemistry 40, 2410–2418 (2001).

    CAS  PubMed  Google Scholar 

  143. 143.

    Viviani, V. R., Arnoldi, F. G. C., Ogawa, F. T. & Brochetto-Braga, M. Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey. Luminescence 22, 362–369 (2007).

    CAS  PubMed  Google Scholar 

  144. 144.

    Modestova, Y., Koksharov, M. I. & Ugarova, N. N. Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence. Biochim. Biophys. Acta 1844, 1463–1471 (2014).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank New York University Abu Dhabi for financially supporting this work through the Research Enhancement Fund scheme (project “Red- and Green-Emitting Luciferases: Determination of the Color Emission Mechanism”). This work was also supported by the Human Frontier Science Program (project RGY0081/2011, “Excited-State Structure of the Emitter and Color-Tuning Mechanism of the Firefly Bioluminescence”).

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C.C.-L. and P.N. contributed to conceptualization, researching, analysis, discussions, writing the original draft and review/editing of the final draft. N.M.L. contributed to researching, analysis, discussions and review/editing of the final draft. S.S. contributed to analysis, discussions and review/editing of the final draft.

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Correspondence to Panče Naumov.

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Carrasco-López, C., Lui, N.M., Schramm, S. et al. The elusive relationship between structure and colour emission in beetle luciferases. Nat Rev Chem 5, 4–20 (2021). https://doi.org/10.1038/s41570-020-00238-1

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