Photochemical & Photobiological Sciences PAPER Cite this: Photochem. Photobiol. Sci., 2020, 19, 313 Received 6th November 2019, Accepted 27th January 2020 DOI: 10.1039/c9pp00436j rsc.li/pps The interaction of C-terminal Tyr208 and Tyr13 of the first α-helix ensures a closed conformation of ctenophore photoprotein berovin Ludmila P. Burakova, Elena V. Eremeeva and Eugene S. Vysotski * Light-sensitive Ca 2+ -regulated photoprotein berovin is responsible for the bioluminescence of the cteno- phore Beroe abyssicola. It shares many properties of hydromedusan photoproteins although the degree of identity of its amino acid sequence with those of photoproteins is low. There is a hydrogen bond between C-terminal Pro and Arg situated in the N-terminal α-helix of hydromedusan photoproteins that supports a closed conformation of the internal cavity of the photoprotein molecule with bound 2-hydro- peroxycoelenterazine. The C- and N-terminal hydrogen bond network is necessary to properly isolate the photoprotein active site from the solvent and consequently to provide a high quantum yield of the bioluminescence reaction. In order to find out which berovin residues perform the same function we modified the N- and C-termini of the protein by replacing or deleting various amino acid residues. The studies on berovin mutants showed that the interaction between C-terminal Tyr208 and Tyr13 localized in the first α-helix of the photoprotein is important for the stabilization and proper orientation of the oxy- genated coelenterazine adduct within the internal cavity as well as for supporting the closed photoprotein conformation. We also suggest that the interplay between Tyr residues in ctenophore photoproteins occurs rather through the π–π interaction of their phenyl rings than through hydrogen bonds as in hydro- medusan photoproteins. Introduction Ca 2+ -regulated photoproteins are responsible for the light emission of various marine organisms such as cnidarians and ctenophores and consist of a single (around 20–25 kDa) poly- peptide chain. The protein inner cavity contains a molecule of 2-hydroperoxycoelenterazine, a noncovalently bound oxyge- nated coelenterazine derivative. 1,2 This particular structural feature distinguishes Ca 2+ -regulated photoproteins from other bioluminescent enzymes, since their light emission becomes independent of the presence of molecular oxygen in the reac- tion mixture. However, oxygen is involved in photoprotein bio- luminescence by its necessity to form an active photoprotein from apophotoprotein and coelenterazine under Ca 2+ -free conditions. 3,4 Photoprotein bioluminescence is triggered by Ca 2+ binding which induces a decarboxylation of the bound 2-hydroperoxy adduct of coelenterazine and the subsequent generation of protein-bound coelenteramide in its excited state. Its relaxation to the ground state is accompanied by light emission with λ max at 469–495 nm, depending on the origin of the protein. 5 Calcium is considered to be an allosteric modu- lator which speeds up the rate of photoprotein bio- luminescence reactions, because even without calcium ions all photoproteins display a very low level of light emission called the “calcium-independent luminescence”. 6 After Ca 2+ binding to photoprotein its light intensity increases 1 million-fold or more. Ctenophores (comb jellies) are a phylum of invertebrate animals that dwell in sea waters worldwide and practically all species of the group, with a rare exception, are bioluminescent. 7,8 The bright bioluminescence of ctenophores is also conditioned by the Ca 2+ -regulated photoproteins. By now, several cDNA genes encoding Ca 2+ -regulated photoproteins from different cteno- phores have been cloned. These are berovin from Beroe abyssi- cola, 9 bolinopsin from Bolinopsis infundibulum, 10,11 mnemiopsin from Mnemiopsis leidyi, 12,13 and bathocyrovin from Bathocyroe fosteri. 14 The comparison of amino acid sequences of cnidarian photoproteins with those determined for ctenophore photopro- teins showed only 29.4% degree of identity. 9,14 At the same time both types of photoproteins have three Ca 2+ -binding sites consist- ing of 12 canonical residues typical of EF-hand Ca 2+ -binding pro- teins. 15 Although amino acid sequences of cnidarian and cteno- phore photoproteins are different, many features of these pro- Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, Russia. E-mail: eugene.vysotski@gmail.com; Fax: +7 (391) 290-54-90; Tel: +7 (391) 2494430 This journal is © The Royal Society of Chemistry and Owner Societies 2020 Photochem. Photobiol. Sci. , 2020, 19, 313–323 | 313