DOI: 10.1002/cptc.201700060 Communications

Investigation of the Mechanism: Chemically Initiated Electron Exchange Luminescence or Twisted Intramolecular Charge Transfer? Phong D. Ngo and Steven O. Mansoorabadi*[a]

Ubiquitous in the world’s oceans, are capable of fantastic displays of bright-blue bioluminescence. This lumi- nosity is a consequence of the oxidation of an open-chain , dinoflagellate luciferin (LH2), by the dino- flagellate (LCF). While many other bioluminescence systems are well understood, the reaction mechanism of LCF remains enigmatic. A comprehensive density functional theory investigation was used to evaluate several competing mecha- nisms of LCF catalysis employing distinct excited-state lumino- phores. The results provide strong evidence in favor of a mech- anism of dinoflagellate bioluminescence involving an excited- state gem-diol(ate) intermediate. Analysis of the molecular orbitals relevant to the emission process indicates that catalysis from the E isomer of LH2 is likely to proceed via a chemically initiated electron-exchange luminescence reaction, whereas Figure 1. Calculated UV/Vis absorption spectral changes for the conversion that from the Z isomer may involve the formation of a biologi- of the chromophore (highlighted in blue) of the E isomer of dinoflagellate cally unprecedented twisted intramolecular charge transfer luciferin (LH2) to oxyluciferin (LO). state.

minescence (CTIL) pathways, where intramolecular charge Dinoflagellates are an important group of eukaryotic microor- transfer induces OÀO bond cleavage, the liberation of ganisms found in fresh water and marine environments.[1] Cer- dioxide, and the concomitant formation of the excited-state tain species of dinoflagellates are photosynthetic and capable oxyluciferin (LO) luminophore that relaxes with the emission of of bioluminescence induced by physical agitation.[2] The biolu- visible .[15,16] However, unlike (and coelenterate) bio- minescence reaction, which is regulated on a circadian rhythm, luminescence, the reaction catalyzed by LCF does not proceed involves the oxidation of an open-chain tetrapyrrolic substrate, with the formation of carbon dioxide;[5] thus, LCF is likely to dinoflagellate luciferin (LH2), by the enzyme dinoflagellate luci- utilize a novel bioluminescence mechanism. ferase (LCF) as illustrated in Figure 1.[3,4] Of all the major classes Two main computational methodologies have been em- of luciferase (i.e. firefly, bacterial, coelenterate, and dinoflagel- ployed in the investigation of firefly bioluminescence: multi- late), least is known about the mechanism of light production configurational methods, such as complete active space self- by dinoflagellate luciferase.[5] consistent field (CASSCF) and multiconfigurational second- The firefly bioluminescence mechanism has been extensively order perturbation theory (CASPT2), and time-dependent den- investigated using computational chemistry, and key insights sity functional theory (TD-DFT) methods.[17–19] The multiconfi- into the structure of the bioluminophore have been ob- gurational approach can achieve high accuracy while being ap- tained.[6–14] During the catalytic cycle of , a diox- plied to systems with degenerate electronic states (for example etanone intermediate is formed, which is thought to decay at conical intersections). However, these methods can be com- through either stepwise chemically initiated electron exchange putationally expensive and require a careful choice of the luminescence (CIEEL) or concerted charge transfer induced lu- number of electrons and orbitals in the active space, and thus require a preliminary understanding of the relevant excited states. In contrast, TD-DFT methods are relatively inexpensive [a] Dr. P. D. Ngo, Prof. S. O. Mansoorabadi Department of Chemistry and Biochemistry and can be readily applied to large systems. However, conven- Auburn University tional DFT functionals often perform poorly when applied to 179 Chemistry Building excitations involving long-range charge transfer (such as Ryd- Auburn, AL 36849 (USA) berg transitions). More recently, TD-DFT functionals employing E-mail: [email protected] a long-range correction scheme have been developed with im- Supporting Information and the ORCID identification number(s) for the author(s) of this article can be found under: proved performance for the calculation of charge-transfer https://doi.org/10.1002/cptc.201700060.

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states, and have been successfully applied in the investigation was proposed, wherein an excited state LO intermediate trans- [12, 13,20] of the firefly bioluminescence system. fers to a second LH2 molecule (or an analogue thereof), To gain insight into the mechanism of dinoflagellate biolu- which serves as the bioluminophore.[28] This proposal is based

minescence, a computational investigation utilizing TD-DFT on the fact that, unlike other bioluminescent systems, the LH2 was employed. Ground- and excited-state for key in- substrate, rather than the LO product, is fluorescent.[4,28] Al- termediates in several proposed mechanisms of LCF catalysis though the LCFs from most dinoflagellates contain three ho-

were calculated using the long-range and dispersion-corrected mologous catalytic domains (and thus three LH2-binding sites) hybrid functional wB97X-D and the 6–311++G(d,p) basis set within a single polypeptide, it is unclear whether these active as implemented in the Gaussian 09 software package.[21,22] This sites are in close enough proximity to allow for efficient computational scheme was chosen because it was found to energy transfer.[29] In addition, the structure and properties of perform well in the calculation of low-lying excited states of the putative luciferin analogue were not described and poten- [23] organic molecules. tial bioluminophores derived from the reaction of LH2 with To verify the performance of the chosen method while re- molecular were not considered.[28] ducing computational cost, calculations were performed on It has been proposed that the LCF catalytic cycle involves

the LH2 chromophore synthesized by Stojanovic and Kishi deprotonation of the E ring of LH2 by an active site base (for [24,25] (Figure 1). The synthetic LH2 chromophore was shown to example E1105 in the structure of LCF from Lingulodinium pol- abs have very similar UV/Vis absorption (lmax =389 nm) and fluo- yedrum) to generate an enolate anion that reacts with molecu- fl abs rescence (lmax =466 nm) properties to full-length LH2 (lmax = lar oxygen to form a hydroperoxide intermediate, the excited fl [4,24] 388 nm, lmax = 474 nm). Moreover, the LH2 chromophore state of which could serve as the bioluminophore by relaxing was found to be a luminescent substrate of LCF, being con- with radiative emission of blue light at 474 nm.[4,24, 25,30] Howev- abs verted into the chromophore of LO (lmax =340 nm) with the er, several alternative reaction mechanisms can also be envi- concomitant emission of light.[4,24] Thus, a reduced model con- sioned wherein the light-emitting species is an excited-state

sisting of the chromophore of LH2 should retain all the neces- peroxy anion, gem-diol, or gem-diolate intermediate (Figure 2). sary structural features to form the bioluminophore. The configuration of the double bond connecting the E and

D rings in LH2 has not been conclusively assigned. LH2 is a ca- tabolite of a, which has the Z configuration about this bond.[26] In contrast, comparison of the 13C NMR chemical shifts of the two isomers of the synthesized chromophore with

that of purified LH2 suggests that the bioluminescent substrate contains the E configuration.[24] However, the Z isomer of the

synthesized LH2 chromophore was found to readily isomerize to the more stable E form, and it is unclear whether full-length

LH2 retains its configuration during purification from dinofla- gellate cell extracts.[24] Therefore, calculations were performed

on both isomers of the LH2 chromophore. The calculated UV/Vis absorption spectra for the conversion of the E isomer of LH into LO are shown in Figure 1 and repro- Figure 2. Chromophores of possible intermediates in the dinoflagellate luci- 2 ferase (LCF) catalytic cycle, whose excited states may serve as the biolumino- duce well the experimental trend observed for the enzymatic phore. reaction in 0.2m phosphate buffer, pH 6.3.[4] In these calcula- tions, the solvent (water) was implicitly included using the po- larizable continuum model (PCM).[27] The calculated vertical ex- To ascertain the feasibility of these competing mechanisms

citation wavelengths for the E and Z isomers of LH2 (lex = and shed light on the structure of the potential biolumino-

322 nm and 320 nm, respectively) and LO (lex =289 nm and phore, the geometries of the ground and first excited state of 288 nm, respectively) are similar in magnitude and underesti- each of these intermediates were optimized and the corre- abs mate the corresponding experimental absorption lmax value. sponding vertical excitation and emission wavelengths were Likewise, the calculated vertical emission wavelengths from calculated using the computational methodology described

the first excited state of the E and Z isomers of LH2 (lem = above. The relative energy of each species along the reaction 405 nm and 415 nm, respectively) are smaller than the experi- coordinate was then determined by comparison with the em mental fluorescence lmax value. A similar underestimation of ground-state energies of the corresponding LH2 substrate

the experimental absorption and emission wavelengths of LH2 (+O2) and LO product (+H2O). These calculations also included by 50–70 nm was obtained using the Coulomb-attenuated acetate/acetic acid as a mimic of the putative active site acid/ hybrid exchange-correlation functional (CAM-B3LYP) in a very base residue as appropriate. The effects of surroundings (aque- recent computational investigation of dinoflagellate biolu- ous versus proteinaceous environment, the latter modeled minescence.[28] In that study, the spectroscopic properties of using a dielectric constant of 4) and stereochemistry/conforma-

a slightly larger model of LH2 and LO were examined and tion of the O2-derived functional groups (R versus S/eclipsed a mechanism of LCF catalysis involving Dexter energy transfer versus staggered, as applicable) on the intermediate energies

& ChemPhotoChem 2017, 1,1–6 www.chemphotochem.org 2 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ÝÝ These are not the final page numbers! Communications and emission wavelengths were also examined (see the Sup- The ground-state energies of the Z isomers of the gem-diol porting Information for full computational results). (À83.6 Æ0.1 kcalmolÀ1) and gem-diolate (À65.7 Æ0.4 kcal In the case of the peroxy anion intermediate, the calculated molÀ1) intermediates are very similar to those of the corre- vertical emission wavelength (lem) from the first excited state sponding E isomers. In contrast, the first excited state of the typically lies between 548–610 nm, with a weak oscillator gem-diol (À25.3 Æ0.2 kcalmolÀ1) and gem-diolate (À9.6Æ strength (f) ranging from 0.005–0.019. In certain conformations 0.1 kcalmolÀ1) intermediates are much lower in energy than of the E isomer of the peroxy anion intermediate, the pyrrole the corresponding E isomers. Interestingly, the large discrepan- of the D ring donates a bond to the termi- cy in the calculated energies of the first excited state of the nal oxygen of the peroxide moiety, which increases the intensi- two isomers can be attributed to the fact that, during the ge- ty of the emission and decreases its wavelength to between ometry optimization, the E isomer maintained its planar con- 502–528 nm. This highlights the potential of the enzyme’s formation (likely due in part to an intramolecular hydrogen active site to tune the bioluminescence emission wavelength bond between the E ring carbonyl and the D ring nitrogen), and intensity using hydrogen-bonding interactions. However, while an unexpected rotation occurred about the C15ÀC16 considering that the calculated lem value is likely to be under- bond of the Z isomer such that the E and D rings became estimated, this transition cannot explain the bioluminescence perpendicular to one another. emission of LCF. Moreover, although formation of the ground- The emission wavelengths from the first excited state of the state peroxy anion intermediate from the E isomer of LH2 is on twisted gem-diol and gem-diolate intermediates are far too average energetically favorable (À5.4Æ0.3 kcalmolÀ1), its for- long to be consistent with the bioluminescence process; more- mation from the Z isomer is not (2.2Æ0.5 kcalmolÀ1), and the over, these states are dark with f=0. However, the calculations excited-state peroxy anion is likely to be thermodynamically in- indicate that there is also a second low-lying excited state for accessible in both cases (66.9Æ1.4 kcalmolÀ1). Thus, an excit- each of these intermediates. For the twisted gem-diol inter- ed-state peroxy anion was ruled out as a potential biolumino- mediate, the transition wavelength from this second excited phore. state is between 355–359 nm (with f values varying between The blue-shift in the emission wavelength of the peroxy 0.12–0.16), whereas that for the twisted gem-diolate intermedi- anion intermediate towards the bioluminescence emission ate is between 457–486 nm (with f values from 0.084–0.14). maximum induced by intramolecular hydrogen bonding sug- This suggests that a gem-diolate (or hydrogen-bonded gem- gests that an excited-state hydroperoxide intermediate may be diol) intermediate could also be the bioluminophore if LH2 has a good candidate for the bioluminophore. Indeed, the calculat- the Z configuration. Attempts were made to optimize the ed vertical emission wavelength of the hydroperoxide inter- structure of the second excited state of the twisted gem-diol mediate is much closer to the experimental bioluminescence and gem-diolate intermediates and calculate the vertical emis- emission maximum, ranging between 438–482 nm, with an os- sion wavelength directly. However, these calculations failed to cillator strength of 0.19–0.32. In terms of energetics, formation converge. of the ground-state hydroperoxide intermediate is significantly The data are therefore most consistent with a mechanism of more favorable than that of the peroxy anion (À17.4 Æ1.8 kcal LCF catalysis in which an excited-state gem-diol(ate) intermedi- À1 mol ). However, the excited state energy for the hydroperox- ate serves as the bioluminophore. If the LH2 substrate contains ide intermediate is also quite high (55.6Æ2.0 kcalmolÀ1), and the E configuration about the C15ÀC16 double bond, planarity thus similar concerns apply to the feasibility of its formation. of the E and D rings is likely maintained throughout the cata-

The calculated lem value of the E isomer of the gem-diol in- lytic cycle and the bioluminophore is predicted to be a gem-di- termediate is similar in magnitude to the hydrogen-bonded olate or hydrogen-bonded gem-diol intermediate. Alternatively, peroxy anion (507–512 nm), although the oscillator strength is if LH2 has the Z configuration, the reaction may involve a rota- larger (0.090–0.096). However, in stark contrast to the peroxy tion about the C15ÀC16 bond and the formation of a twisted anion, the formation of both the ground state (À84.8 Æ0.1 kcal excited state gem-diol(ate) intermediate. molÀ1) and excited state (À14.2 Æ0.2 kcalmolÀ1) of the E-gem- The molecular orbitals (MOs) participating in the relevant diol intermediate is favorable. Deprotonation of one of the hy- emission process for the E isomer of the gem-diol and the droxy groups of the E-gem-diol to form the E-gem-diolate inter- Z isomer of the gem-diolate intermediates are shown in mediate results in a blue-shift of the lem value (433–446 nm) Figure 3. In the former case, an electron is transferred from an and a significant increase in oscillator strength (0.22–0.23). orbital centered predominantly on the E ring carbonyl to the However, there is a corresponding increase in the energy of delocalized p-system of the chromophore (Figure 3A). The both the ground state (À67.3 Æ0.3 kcalmolÀ1) and the excited MOs for the analogous transition in the E-gem-diolate inter- state (6.6Æ 0.8 kcalmolÀ1). Regardless, the excited state of the mediate are similar. Thus, if an excited-state E-gem-diol(ate) in- E-gem-diolate intermediate should be readily accessible. There- termediate is to serve as the bioluminophore, an open-shell fore, considering both the relatively low energy of the excited singlet containing an unpaired electron in each of these orbi- state and the magnitude of the emission wavelength, a gem- tals must be formed during the catalytic cycle, and photon diolate (or a hydrogen bonded gem-diol intermediate) inter- emission occurs with intramolecular electron transfer from the mediate is a strong candidate for the bioluminophore if LH2 carbonyl to the conjugated p-system. has the E configuration. In contrast, the transition for the twisted gem-diolate inter-

mediate derived from the Z isomer of LH2 involves the transfer

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At this point the two mechanisms diverge. For the E isomer (Figure 4A), dioxetanolate ring-opening is initiated by electron transfer from the conjugated p-system. A subsequent or con- certed deprotonation of the remaining E ring hydrogen by the adjacent oxyanion generates the excited-state gem-diol(ate) in- termediate and back electron transfer leads to photon emis- sion. Such a mechanism describes a chemically initiated elec- tron-exchange luminescence (CIEEL) process, which has been invoked to explain other known bioluminescent reactions, most notably that catalyzed by firefly luciferase.[5,31] For the Z isomer (Figure 4B), deprotonation of the E ring leads to OÀO bond cleavage and induces a rotation about the Figure 3. Relevant molecular orbitals in the bioluminescence emission pro- C15ÀC16 bond, generating the open-shell singlet excited state. cess for the A) E isomer and B) Z isomer of the gem-diol(ate) intermediate. D to E ring charge transfer is then accompanied by photon emission. This mechanism describes the formation of a twisted intramolecular charge transfer (TICT) state. TICT states have of an electron from an orbital centered primarily on the been implicated in a number of photochemical processes, but C16 atom of the D ring to a MO on the orthogonal E ring (Fig- have yet to be observed in a bioluminescence system.[32] ure 3B). Therefore, if an excited-state Z-gem-diol(ate) is the bio- The mechanisms of LCF catalysis proposed herein (Figure 4) luminophore, an open-shell singlet with one unpaired electron are in contrast to the mechanism involving Dexter energy on each ring of the twisted intermediate is formed during the transfer that was proposed in the recent theoretical study of reaction and light is emitted with charge transfer from the dinoflagellate bioluminescence by Wang and Liu.[28] In that

D to the E ring. study, the experimental observation that LH2 (as opposed to

Mechanisms of LCF catalysis consistent with the above infor- LO) is fluorescent, with a lem value similar to the biolumines- mation are shown in Figure 4. Starting from either isomer of cence emission maximum, was confirmed using TD-DFT.[28] On

LH2, the catalytic cycles involve deprotonation of the E ring by this basis, it was suggested that an excited-state LO intermedi- the active site base to generate an enolate anion that reacts ate formed during the LCF catalytic cycle could transfer energy

with molecular oxygen to form a dioxetanolate intermediate. to a second LH2 molecule (or an analogue thereof), which would then serve as the bioluminophore by relaxing with the radiative emission of light.[28] The data presented herein sug- gest that an excited-state intermediate derived from the reac-

tion of LH2 with molecular oxygen (that is, an excited state gem-diol(ate) intermediate) can directly serve as the lumino- phore in dinoflagellate bioluminescence. In summary, TD-DFT was used to calculate the ground- and excited-state energies of luminophores from several proposed mechanisms of LCF catalysis. Comparison with the experimen- tal bioluminescence emission wavelength and an evaluation of the thermodynamic feasibility of forming each species favors a gem-diol(ate) intermediate over a peroxy anion or hydroper- oxide as the bioluminophore. If the LCF catalytic cycle begins

with the more stable E isomer of LH2, the reaction is likely to

involve a CIEEL mechanism. However, if LH2 has the Z configu- ration, the calculations suggest that a twisted excited state gem-diol(ate) intermediate could serve as the bioluminophore and LCF would catalyze a biologically unprecedented TICT reaction.

Acknowledgements

This work was supported by the National Science Foundation CAREER Award CHE-1555138 (from the Division of Chemistry) and made possible in part by a grant of the high performance computing resources and technical support from the Alabama Figure 4. Proposed mechanisms of dinoflagellate luciferase (LCF) catalysis in- Supercomputer Authority. volving A) chemically initiated electron exchange luminescence (CIEEL), and B) twisted intramolecular charge transfer (TICT).

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P. D. Ngo, S. O. Mansoorabadi* Sea how they glow: A comprehensive DFT investigation was used to evaluate && – && several competing mechanisms for di- Investigation of the Dinoflagellate noflagellate bioluminescence, a conse- Bioluminescence Mechanism: quence of the oxidation of dinoflagel-

Chemically Initiated Electron late luciferin (LH2) by the enzyme dino- Exchange Luminescence or Twisted flagellate luciferase to form oxyluciferin Intramolecular Charge Transfer? (LO). Strong evidence in favor of a mech- anism involving an excited-state gem- diol(ate) intermediate is presented.

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