Molecular mechanism of Symplectoteuthis bioluminescence—Part 4: Chromophore exchange and oxidation of the cysteine residue

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Abstract

Symplectin is one of the few photoproteins, which forms covalent bonds with the dehydro-coelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH’s of the cysteine residues via conjugate addition reaction. This photoprotein contains the chromophore molecules at the binding cites first, and then moves to the active cite Cys-390 for the luminescence. The current study focuses on these dynamic aspects of the chromophore using the natural photoprotein by analyzing the fluorescence changing of the DCL chromophores analogs with 8-(4′-methoxyphenyl)- or 8-(2′-naphthyl)-group and 2-(2′,4′-difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at slightly acidic media and also from the luminescence function observed at the optimum pH 7.8. The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid chromatographic detection from the spent solutions.

Introduction

This article is the continuation of Part 3 of our previous report on Symplectoteuthis bioluminescence.1

Light emission from living cells has been commonly used in chemical biology research as one of the important tools for specific visualization of the target molecules on the basis of molecular-binding in living cells. Coelenterazine is the most typical chromophore in marine bioluminescent systems, and recently 2-hydroperoxycoelenterazine is the key feature in the calcium dependent photoproteins such as aequorin and obelin. These studies have made the molecular mechanism of the bioluminescence by means of structural biology using X-ray analysis of the co-crystallized photoproteins with the substrates.2 We have been working on the coelenterazine-containing photoprotein symplectin, the luminescence of which is triggered by monovalent ions such as Na+ and K+.3 This photoprotein symplectin works in an oceanic squid, Symplectoteuthis oualaniensis (Japanese name, TOBI-IKA) collected in the sea around Okinawa, Japan,4 and obtainable in a water-insoluble fraction but only soluble with high salt-concentration, for example, KCl >0.6 mol/L.4 In 2008, the amino acid sequence of symplectin was elucidated with its 501 AA containing 11 cysteine residues (including 3 Cys-S-S-Cys). The current study was carried out using the purified photoprotein from the squid since gene-manipulated preparation of this protein has not been reported. Isobe et al. assigned that the 390-Cys worked as the active site of symplectin by means of LC–MS-detection and the following experiments.5 Namely, the chromo-peptide fragment, chrom-S-390C-G-L-393K, was observed by using the mono-fluorinated dehydro-coelenterazine analog; thus, a peak corresponding to MW 843.55 as the evidence of the coelenterazine analog being bound to the active site before the luminescence. Another peak equivalent to MW 831.53 was observed after the luminescence from the tryptic digest as the coelenteramide analog.5 This detection system includes a house-modified capillary-HPLC connected to ESI-Q-TOF/MS on the samples from the photoprotein, which was reconstructed with synthetic chromophore and then hydrolyzed with proteolytic enzyme such as Trypsin or Lys-C.5 Further proof of the active site was implemented using di-fluorinated coelenterazine, which binds to the same 390-Cys residue more tightly. It gave the molecular ions of the chromopeptide after luminescence at m/z 849.3 as well as the fragment ions (m/z 735, 588, 419, 306 and 278) from the capillary HPLC-ESI-Ion Trap MS to show the coelenteramide substructure.1

Shimomura states that many marine bioluminescent animals give light by using coelenterazine or similar compounds as the substrate having common chromophore.6 The oxidation mechanism and peroxy intermediates of these chromophores show the common substructures as the hydroperoxide 5 and peroxylactone (dioxetanone) 6,6 as has been demonstrated by Isobe and co-workers to prove the more detailed mechanism in a direct manner using 100%-13C-enriched luminous coelenterazine analogs at some specific carbon atoms and analyses through low-temperature 13C NMR of the short-life time peroxidic intermediates (Fig. 1).7 The presence of 2-hydroperoxycoelenterazine in aequorin and obelin has been reported recently.2, 8

In this case, those reactive intermediates were prepared via low-temperature photo-oxygenation in CF3CD2OD solvent. The NMR at −78 °C proved the structures of 2-hydroperoxy-coelenterazine analog 5 and dioxetanone 6 with concomitant measurements of luminescent spectra from these intermediates under different media. Namely, 5 and 6 provided significant luminescence spectra derived not only from neutral media but also even from acidic media by simply allowing the temperature at ca. −50 °C or 0 °C, respectively. This provided new tools elucidating the excited-molecular species, which is strong contrast to the fact that previous chemi-luminescence had been reported only under strongly basic conditions in dipolar aprotic solvents such as DMSO. Titrative reduction of the peroxides 5 and 6 at −78 °C with triphenylphosphine or diphenyl sulfide took place in the stoichiometry-dependent manner with diminishing the amount of luminescence; (triphenylphosphine was as example as shown in Fig. 2).7 The products in the reduction were amino-pyrazines 9 and triphenylphosphine oxide or diphenyl sulfoxide. So the bioluminescence mechanism should include the same chemical principle on the enzymic surface. Recently, Isobe et al. further reported the fact that two different bioluminescence systems including sulfur atom of DTT (dithiothreitol) attached at the cysteine residues of the photoprotein aequorin.5, 9 Involvement of the sulfur atom of Cys residues provided new tools for the studies of bioluminescence, which is described in the current study.

Kongjinda and Isobe1 reported that two di-fluoro-dehydrocoelenterazine regio-isomers (FF-DCL-bn: deep red color, non-fluorescent) 12 and 13 showed quite different bioluminescence behavior in symplectin photoprotein from that of natural chromophore 11 (4-OH-DCL-bn). Namely, 2,4-difluoro-analog 12 (2,4-FF-DCL-bn) provides larger amount of the luminescent light than the natural chromophore 11, while 2,6-difluoro-analog 13 (2,6-FF-DCL-bn) gives smaller amount (1/3–1/4) of light from 11.9 Both of these two chromophores 12 and 13, however, showed similar behavior such as increase of the green fluorescence at 520 nm during the incubation to apo-symplectin at pH 6.0 in accordance with the binding as a conjugate addition manner of the SH group of Cys to these chromophores (Fig. 5). When the pH of the DCL-analog-incubated photoproteins was changed to pH 7.8, both analogs show decreasing of the green fluorescent intensities. Namely, the non-bioluminescent species from 13 was consumed as well like the luminescent one 12. This suggested some different pathways might take place for each of them on the protein surface when subjected to the luminescence conditions.

The synthetic route of the coelenterazine-chromophores10 was redesigned to avoid using hazardous diazo-reagents as reported by Shimomura and Kishi11 and the improved version reported by Kondo et al.12 Alternative routes on the basis of Pd-mediated cross coupling13 was also developed by Makarasen et al.,14 and Phakhodee and Isobe.15 Chou, one of the authors of this paper, has published a new synthetic route from aminopyrazine, which was suitable for the synthesis of the current study.16 In this paper, we prepared such chromophores 1117 (see Fig. 3) via the new synthetic route that have different fluorescence maxima and intensities emerging after binding with the cysteine residues. These compounds led us to have the first experimental achievement of the chromophore-exchange on the photoprotein, symplectin, from originally bound-natural chromophore 11 to the new difluoro-derivatives 12, 13, 15, and 16. Of particular interests, 15 (2,4-FF-DCL-nap) and 16 (2,6-FF-DCL-nap) derivatives did not exhibit strong fluorescence at 520 nm. We have confirmed these observations with symplectin by using various sample preparations which yield different amounts of apo-symplectin.

Section snippets

Preparation of the symplectin solution

A partially purified (SDS–PAGE single band) sample solution of symplectin was prepared on the basis of its limited solubility upon KCl-concentration according to our previously reported method with slight modification.1 Every symplectin-preparation was prepared from the frozen-photogenic organ by cutting as a round disk by using a 1.5 cm-diameter borer to give a disk-weight of 50 mg (±2 mg). The disk was homogenized with a glass-homogenizer using the basic buffer (containing 0.4 M KCl, pH = 7.8, 1000 

Conclusions

The fluorescence change by the incubation to symplectin provided us a good evidence to prove the chromophore, which bound to the binding sites of symplectin, can exchange with the outcome synthetic chromophore depended on the relative binding affinity to cysteine residue. Because 19 (Cys-2,4-FF-DCL-nap) only shows weak fluorescence at 600 nm, this characteristic nature makes the observation much easier to confirm the decreasing of the fluorescence at 520 nm, which is represented by

Experimental

Instruments: Fluorescence spectra were measured with a JASCO FP-2020+ spectrometer and Hitachi F-4500 spectrometer. Bioluminescence was obtained on a Hamamatsu Photonic Multi-channel Analyzer, PMA-11 by simultaneous integration of light with different wavelengths (300–600 nm) without scanning against the wavelength. Total amounts of the luminescent light were acquired with Gene Light GL-200S to obtain the relative light yield. Centrifugation was performed by using a Hettich Mikro 120 centrifuge

Acknowledgments

Financial support from Ministry of Science and Technology (the previous name is National Science Committee in Taiwan) is gratefully acknowledged. Authors are also indebted to National Tsing Hua University for the equipments.

References and notes (16)

  • F.I. Tsuji et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (1981)
  • M. Isobe et al.

    Proc. Jpn. Acad., Ser. B

    (2008)
  • S. Inoue et al.

    Chem. Lett.

    (1975)
  • O. Shimomura et al.

    Biochem. J.

    (1989)
  • A. Makarasen et al.

    Bull. Chem. Soc. Jpn.

    (2009)
  • V. Kongjinda et al.

    Chem. Asian J.

    (2011)
  • E.V. Eremeeva et al.

    Photochem. Photobiol. Sci.

    (2014)
    E.V. Eremeeva et al.

    J. Photochem. Photobiol. B: Biol.

    (2013)
  • H. Takahashi et al.

    Bioorg. Med. Chem. Lett.

    (1993)
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