Stabilizing mechanisms of three possible isomers (phenolate-keto, phenolate-enol, and phenol-enolate) of the oxyluciferin anion hydrated with quantum explicit water molecules in the first singlet excited state were investigated using first-principles Born–Oppenheimer molecular dynamics simulations for up to 1.8 ns (or 3.7 × 106 MD steps), revealing that the surrounding water molecules were distributed to form clear single-layered structures for phenolate-keto and multi-layered structures for phenolate-enol and phenol-enolate isomers. The isomers employed different stabilizing mechanisms compared to the ground state. Only the phenolate-keto isomer became attracted to the water molecules in its excited state and was stabilized by increasing the number of hydrogen bonds with nearby water molecules. The most stable isomer in the excited state was the phenolate-keto, and the phenolate-enol and phenol-enolate isomers were higher in energy by ∼0.38 eV and 0.57 eV, respectively, than the phenolate-keto. This was in contrast to the case of ground state in which the phenolate-enol was the most stable isomer.

Firefly bioluminescence, or luciferin–luciferase reactions that emit a photon with high quantum efficiency,1–3 is one of the most familiar wonders in nature and has attracted both scientific and industrial interest. One of the most lasting enigmas of this system is why the emission color of the light emitter oxyluciferin in vitro bioluminescence assays changes in response to various environmental factors including temperature, pH of the solvent, presence of bivalent metals, and protein mutations.1–10 Oxyluciferin has a number of known conjugate forms, including two neutral isomers (phenol-keto and phenol-enol), three anionic isomers (phenolate-keto, phenolate-enol, and phenol-enolate), and a dianion isomer (phenolate–enolate). Various color-change theories have been proposed and evaluated in the past, including the mono-dianion theory, C2C2′ bond theory, keto–enol equilibrium theory, resonance theory, influence of protein micro-environments, and hydrogen bonds.7,11–15 Notable progress has been made recently with the application of systematic chemical spectroscopy experiments, which have been able to circumvent oxyluciferin instability. These experiments have identified the photoabsorption and photoemission spectra for each isomer and the derivative of oxyluciferin in aqueous solutions and confirmed that the phenolate-keto isomer is a red light emitter and that phenolate-enol is a yellow-green light emitter in water.16–18 This indicates that the emission color changes are the result of keto–enol tautomerization in hydration structures. That is, the hydration structures of the surrounding water molecules play an essential role in determining the optical properties and stability of the oxyluciferin isomers.

Many theoretical studies on oxyluciferin using quantum-chemistry methods and molecular dynamics (MD) simulations have been conducted, with these studies being assisted by the significant growth in computational power in the recent past.19–40 Although emission colors and transition energies have been extensively evaluated, there are relatively few studies focused on the stability of different oxyluciferin isomers. This may be because accurate simulations of stability in water have historically been very difficult, and because studies using both vacuum and modeled environments, such as the polarizable continuum solvation model (PCM), always provide a common result, the phenolate-keto form is the most stable in the ground state (S0).26,29 To tackle the challenge of understanding oxyluciferin stability, we created two full-quantum-mechanical first-principles Born–Oppenheimer MD (BOMD) simulations for the oxyluciferin anions hydrated with 64 explicit quantum water molecules [using neither PCM nor molecular mechanics (MM) solutions] in 2016 and 2019.34,39 These simulations each required 335 days of wall clock time for each isomer using 12 INTEL Xeon 6148 processors [240 central processing unit (CPU) cores]. These simulations have revealed, to our surprise, that three oxyluciferin isomers exhibit similar stability profiles and that the phenolate-enol form rather than the phenolate-keto form is the most stable in the ground state S0 in water, where the charge leakage from the oxyluciferin anion to the aqueous solution had a significant effect on its optical property.34 In addition, we were able to show good reproducibility for the experimental absorption spectra in the experiments.39 

To obtain a deeper insight into the stabilizing mechanism of these oxyluciferin anions and to investigate the origin of the emission or fluorescence color changes, which occur at the quasi-thermal-equilibrium points of the electronic excited states,41 it was necessary to complete the same BOMD simulations without the modeled solutions in the first singlet excited (S1) state as well. However, such long-time BOMD calculations on the S1 state were prohibitively heavy for those based on time dependent density functional theory (TDDFT).. For instance, in our previous work of TDDFT/CAM-B3LYP calculations,39 it took more than 4 h to obtain the S1 energy of a single structure for our system, an oxyluciferin ion dissolved in 64 water molecules. Furthermore, these S1 energy calculations were only done with the free boundary surrounded by the PCM. Here, we employ the restricted open-shell Kohn–Sham method (ROKS)42/BLYP43,44-D245 method, which is efficient enough to perform long-time BOMD runs for this system with a full molecular description of the condensed matter under the periodic boundary condition. Based on these considerations, we performed first-principles BOMD for three possible isomers (phenolate-keto, phenolate-enol, and phenol-enolate) of the oxyluciferin anions hydrated with 64 explicit quantum water molecules in the S1 state. To complete our calculations, it was necessary to complete the excited state BOMD analysis for 1.8 ns, corresponding to 3.7 × 106 MD steps. This was done using ∼687 days of wall clock time for each isomer. Our long-time BOMD analyses clearly showed that the surrounding water molecules form a clear single-layered structure for the phenolate-keto isomer and multi-layered structures for the phenolate-enol and phenol-enolate isomers in the excited state. The different hydration mechanisms were confirmed between the oxyluciferin isomers at the first single excited state. While phenolate-keto is more stabilized by attracting water molecules than phenolate-enol and phenol-enolate. The phenolate-keto form was shown to have a lower energy (∼0.4 eV lower) than the phenolate-enol form, making it the most stable isomer in the S1 state.

We applied the first-principles BOMD to the lowest singlet excited state (S1) of the three possible isomers (phenolate-keto, phenolate-enol, and phenol-enolate) of the oxyluciferin anion, put into a cubic unit cell of 13.3723 × 13.3723 × 13.3723 Å3, together with 64 water molecules. The temperature was controlled to be 300 K during the BOMD using the Nose–Hoover algorithm.46,47 The excited state is described by ROKS42 with the BLYP functional,43,44 including van der Waals interactions at the D2 level.45 Other computational details are the same as the BOMD at the ground state. Therefore, a straightforward comparison between the ground and excited state properties is possible. We analyzed all of the converged data after 800 ps and used the averaged values for discussion on the stabilizing mechanism.

Hereafter, we refer to phenolate-keto, phenolate-enol, and phenol-enolate as keto, enol, and enolate. Figure 1 shows the distribution of the water molecules surrounding the oxyluciferin anions in the ground and excited states (note that the figures representing the ground state were made using the molecular geometries described in Ref. 39). There are clear differences in the water molecule distributions between the three isomers and the ground and excited states. In the ground state, the enol, which has five water-rich areas (two of them near the oxygen atom of the benzothiazole moiety and the other three distributed across the oxygen and two nitrogen atoms of the thiazole moiety), seems to be the most hydrophilic isomer as the keto isomer has only four-water rich regions and the enolate isomer has just two (see Fig. 1). We will use the terms hydrophilic and hydrophobic to represent the relative tendency to attract and repel, respectively, the surrounding water molecules. In the excited state, the five water-rich areas of the enol isomer are seen to be much smaller and less distinct than in the ground state, while the keto isomer extends these water-rich areas and makes each of them more distinct. Visualization of the differences between the ground and excited states allows us to observe these changes in more detail (see the right-hand sidein Fig. 1). Notable changes in the distribution of the water molecules between the ground and excited states are observed for both the keto and enol isomers: the keto isomer attracts water molecules around the oxygen and sulfur atoms of the benzothiazole moiety, while the enol isomer not only attracts the water molecules around the same two oxygen atoms as keto but also repels these molecules at the nitrogen atoms. This is less clearly observed for the enolate isomer. Interestingly, the first and second layered structures of the water molecules grow between the ground and the excited state by decreasing randomness. Note that we restrict the present discussion to the plotted area in Fig. 1 because of the limited size of unit cell size. Figure 1 clearly suggests that the differences in the isomers influence the distribution of water molecules over a wider area, and these changes are not restricted to the local distribution of water molecules in the vicinity of the oxyluciferin anion.

FIG. 1.

Distribution of water molecules surrounding (a) keto, (b) enol, and (c) enolate isomers. The color bar representing the rich/poor water distribution is for the six figures on the left with the pink background. The color bar representing the increase/decrease of the S1 − S0 difference is for the three figures on the right with the green background. The translucent orange ellipses in the three left-most figures are shown as guides to the eye to distinguish the single- and the double-water layers confirmed in the S1 excited states. The computational details to obtain these figures are described in supplementary material, S1.

FIG. 1.

Distribution of water molecules surrounding (a) keto, (b) enol, and (c) enolate isomers. The color bar representing the rich/poor water distribution is for the six figures on the left with the pink background. The color bar representing the increase/decrease of the S1 − S0 difference is for the three figures on the right with the green background. The translucent orange ellipses in the three left-most figures are shown as guides to the eye to distinguish the single- and the double-water layers confirmed in the S1 excited states. The computational details to obtain these figures are described in supplementary material, S1.

Close modal

To discuss these observations more quantitatively and to associate these changes with the simulated order of stability, we went on to count the number of water molecules within 3 Å of each atom of the oxyluciferin anion in both the ground and excited states (Fig. 2). Detailed data are provided in supplementary material, S5. In the ground state, the strongest hydrophilic characteristics were observed for the enol isomer, which was surrounded by ∼11.01 water molecules, which was more than 10.73 for the keto (the least hydrophilic isomer) and 10.86 for the enolate isomers. This is reasonable when we consider that the relative stability of the keto and enol isomers is inverted between the vacuum and aqueous models39 (see Fig. 3). The most strongly hydrophilic isomer, enol, also obtained the largest energy gain by forming hydrogen bonds with the surrounding water molecules, with this gain being the smallest for the most weakly hydrophilic isomer, keto, which formed fewer hydrogen bonds. Consequently, enol is more stable in the ground state than keto in aqueous solutions (note that because the enolate isomer is intrinsically much more unstable than the others in the vacuum, the energy gain of the enolate isomer is not sufficient to reverse the stability order).

FIG. 2.

The number of water molecules located within 3 Å of each atom of the oxyluciferin anion.

FIG. 2.

The number of water molecules located within 3 Å of each atom of the oxyluciferin anion.

Close modal
FIG. 3.

The order of stability for the oxyluciferin anions simulated (a) in a vacuum,26 (b) in a PCM solution,29 and (c) in an aqueous solution at the ground (S0)39 and first excited (S1) states.49 

FIG. 3.

The order of stability for the oxyluciferin anions simulated (a) in a vacuum,26 (b) in a PCM solution,29 and (c) in an aqueous solution at the ground (S0)39 and first excited (S1) states.49 

Close modal

The number of water molecules significantly changes in the excited state: the keto isomer is surrounded by 11.26 water molecules, making it the most hydrophilic isomer, while the enol isomer was surrounded by only 10.14 water molecules, making it the least hydrophilic isomer in the excited state. Excited state keto attracts ∼0.5 more water molecules when compared to its ground state, while the other two isomers repel water molecules in their excited state decreasing their interactions by ∼0.2 and 0.9, respectively. This means that only keto isomer gains stability via an increased number of interactions with the surrounding water molecules, while both the enol and enolate isomers lose energy. As a rough estimation, because a single hydrogen bond between the oxyluciferin anion and the water molecule adds ∼0.6 eV,48 the keto isomer gains ∼0.33 eV (=0.53 × 0.6 eV2) in the excited state increasing its stability, while the enol and enolate isomers lose about 0.54 eV and 0.12 eV, respectively, reducing their stability. These energy gains are large enough to determine the order of stability.

The order of stability for the hydrated oxyluciferin anions determined using BOMD was compared with the same values for the vacuum26 and PCM solutions29 (see Fig. 3). For simplicity, we have only used the relative values for keto in the rest of this manuscript. Unlike the PCM solution simulations, our BOMD simulations suggest that the most stable isomer in the ground state is enol; however, the internal energy of the second most stable isomer, keto, is only 0.06 eV higher than that of enol. The most unstable isomer is enolate, which is ∼0.21 eV higher than enol and 0.15 eV higher than keto. Thus, we determined that the order of stability for these isomers in the ground state was enol → keto → enolate,39 while in the first singlet excited state (S1), the order of stability was keto → enol → enolate. The most stable isomer, keto, had 0.38 eV and 0.57 eV less energy than the enol and enolate isomers, respectively. We noticed that the enol–enolate relative stability (or total energy difference) was maintained at ∼0.2 eV for both the ground and excited states and only the relative stability of keto (keto–enol and keto–enolate) changed significantly. This finding suggests that only keto possesses a different stabilizing mechanism underlying its interactions with the water molecules in the excited state and is consistent with our discussion of Figs. 1 and 2, in which we show that only keto attracted water molecules producing single-layered water molecule structures in the excited state.

According to our MD simulations, the keto form, which has 0.38 eV and 0.57 eV less energy than the enol and enolate isomers, is most likely to emit light in aqueous solutions. Based on this insight and the fact that the relative stability of the oxyluciferin anions is determined by their interactions with the surrounding water molecules, we propose a similar stabilizing mechanism for the protein-induced systems as well. The presence of the protein might change the hydrogen-bonding network, thereby reversing the order of stability of these isomers in vivo. This ansatz may be confirmed in future studies by applying the same degree of BOMD simulations for the protein-induced systems.

In summary, we have studied the stability of the oxyluciferin anion in the lowest singlet excited (S1) state in aqueous solution and compared this with the previously reported results for the ground state. We applied first-principles Born–Oppenheimer MD simulations to three possible oxyluciferin anion isomers (phenolate-keto, phenolate-enol, and phenol-enolate) surrounded by 64 water molecules in the lowest singlet excited state as a proof of principle. We analyzed the trajectories obtained in both the excited and ground state MDs in order to clarify the stabilizing mechanism of hydrated oxyluciferin anions. For the phenolate-keto isomer in the S1 state, a single-layered structure of the water molecules was observed. The different stabilizing mechanisms were thus confirmed: only the phenolate-keto isomer became more attractive to the surrounding water molecules, thus gaining more energy via their interactions, while both phenolate-enol and phenol-enolate isomers lost energy due to their increased hydrophobicity. These observations explain the simulated order of stability and the change in the relative stability between the ground and excited states. We confirmed that the phenolate-keto form is the most stable isomer with 0.38 eV less energy than the phenolate-enol isomer and 0.57 eV less energy than the phenol-enolate isomer. This means that the confirmed order of stability for the excited state is phenolate-keto, phenolate-enol, and phenol-enolate, which is different from the ground state where the order of stability is phenolate-enol, phenolate-keto, and phenol-enolate. We hope that the insights obtained in this study could help future studies reveal the underlying light emission mechanisms of oxyluciferin in vitro and aid in the identification of these mechanisms in vivo.

See the supplementary material for a detailed explanation of the analysis, the BLYP total energies, the cumulative average for BLYP total energies, the number of water molecules, and the three-dimensional plot of the distribution of water molecules.

The data that support the findings of this study are available within the article and its supplementary material.

The present long time BOMD simulations have been run on the supercomputer installed at the Institute for Solid State Physics, The University of Tokyo, and the analysis has been performed by using the supercomputer at the Research Institute for Information Technology, Kyushu University. This work was partially supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI Nos. 18H05519, 18H01693, 18K05208, and 20K03784).

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We roughly estimated the single hydrogen bond energy using an oxyluciferin anion and a bonded water molecule, to which the ground state calculation was done with BLYP+D3/6-311G*. The obtained energy was 0.6 eV on average. Although the hydrogen bond energy is dependent on the oxyluciferin anion site and also on isomers, we employed the averaged value in the present discussion. The details are given in S7.

49.

The current discussion with the internal energies does not cause serious problems in the case of the oxyluciferin anions because the only difference in these isomers is the position of the hydrogen atom. According to recent studies, the entropic effect, that is, oscillation of water density, has a strong correlation with the cavity formation at the interface with the hydrophobic molecule.

Supplementary Material