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QM/MM Study of the Formation of the Dioxetanone Ring in Fireflies Through a Superoxide Ion Romain Berraud-Pache, Roland Lindh, and Isabelle Navizet J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00642 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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The Journal of Physical Chemistry

QM/MM Study of the Formation of the Dioxetanone Ring in Fireflies through a Superoxide Ion Romain Berraud-Pache,† Roland Lindh,‡ and Isabelle Navizet∗,† †Université Paris-Est, Laboratoire Modélisation et Simulation Multi Echelle, MSME, UMR 8208 CNRS, UPEM, 5 bd Descartes, 77454 Marne-la-Vallée, France ‡Dept. of Chemistry - Ångström, University of Uppsala, P.O. Box 538, SE-751 21, Uppsala, Sweden E-mail: [email protected]

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Abstract The bioluminescence emission from fireflies is an astounding tool to mark and view cells. However, the bioluminescent mechanism is not completely deciphered, limiting the comprehension of key processes. We use a theoretical approach to study for the first time the arrival of a dioxygen molecule inside the fireflies protein and one path of the formation of the dioxetanone ring, the high-energy intermediate precursor of the bioluminescence. To describe this reaction step, a joint approach combining classical molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations is used. The formation of the dioxetanone ring has been studied for both singlet and triplet states with the help of MS-CASPT2 calculations. We also emphasises the role played by the proteinic environment in the formation of the dioxetanone ring. The results obtained shed some light on an important reaction step and give new insights concerning the bioluminescence in fireflies.

Introduction The bioluminescence phenomenon is one of the topic that dazzle the research community as well as the population. Among the bioluminescent species, fireflies are the most studied both from an experimental and a theoretical point of view 1–3 . Nowadays, it is known how to tune the emission colour of the bioluminescence 4–6 or increase the bioluminescence intensity 7,8 . However, the understanding of the bioluminescence mechanism itself is far from being completely understood.

In fireflies, the bioluminescence is due to the interaction of a protein called luciferase with an organic molecule, the D-luciferin (LH2 ). The generally accepted mechanism of fire-


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fly bioluminescence is shown in Figure 1. During the first step, the protein catalyses the formation of a D-luciferyl adenylate intermediate. Then, the coordination of a triplet dioxygen molecule to the intermediate leads to the formation of a singlet dioxetanone compound. Subsequently, the decomposition of this dioxetanone gives rise to an emissive molecule known as oxyluciferin (OxyLH2 ), which releases the energy by emitting light in the green tone of the visible spectra 2 .

Figure 1: Mechanism of firefly bioluminescence

Recently, some publications have explored the final steps of the mechanism, i.e. the decomposition of the dioxetanone part and the chemical form of the emitting molecule, the oxyluciferin 9,10 . Nevertheless, only few publications have been released on the approach of the dioxygen (O2 ) molecule and the formation of the dioxetanone ring. Theoretical calculations were performed in implicit solvent (PCM) 11,12 and experimental hypotheses date from the 70’s 13–16 .

The two main hypotheses regarding the formation of the dioxetanone ring

give rise to two different mechanisms. In the first one, the α carbon of the ester group of the luciferin is deprotonated by a nearby basic residue. The dioxetanone ring is then formed by the reaction of the newly-form carbanion on the dioxygen molecule 14 (see Figure 2). In the second hypothesis, the same hydrogen is abstracted in a homolytic way by the dioxygen itself leading to the formation of a hydroperoxide species and the presence of radicals. The radicals subsequently react leading to the formation of the dioxetanone ring 13,15 . 3 ACS Paragon Plus Environment


Figure 2: Graphical representation of the two main hypotheses proposed for the coordination of O2 on the Int substrate; on the left side through an oxygen molecule (hypothesis used in this publication); on the right side through a hydroperoxide molecule.

We have decided to focus on the first hypothesis. This approach seems more relevant because there are basic residues close to the carbon of interest that can play the role of the base. The carbon C1 of the intermediate substrate, named Int in this publication (see Figure 3) was deprotonated and the proton put on the His 245, thus becoming Hip 245. The group of Prof. Branchini shows that when the residue His 245 is mutated in Ala or Phe only 20% of the bioluminescence activity is retained 17 . In contrary to BranchiniâĂŹs conclusion, we think that this residue is quite important, counting for 80% of the activity and might plays a direct basic role in the deprotonation of the intermediate. Even if there are others pathways resulting in the bioluminescence emission, His 245 has to play a major role in the bioluminescence mechanism and we wanted in the present study to see if the role of His 245 as a base leads to a reasonable mechanism. In this publication we study the displacement of O2 inside the protein, after the deprotonation of the intermediate by His245, and the formation of the dioxetanone ring by a theoretical approach coupling classical MD and QM/MM calculations. Is the formation of the dioxetanone ring possible starting with a deprotonated Int substrate and a non-protonated dioxygen molecule? What is the path (intermediate species, conformations, spin states) adopted inside the protein with this hypothesis ?


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Computational details We used a crystallographic structure (PDB 4G36) from the group of Prof. Branchini 15,18 . This structure comes from the North American firefly (Photinus Pyralis) and reproduces the conformation of the protein at the beginning of the bioluminescence reaction when the substrate binds to the AMP.

The PDB file 4G36.pdb was downloaded from the RSCB PDB website. The missing loops were added with Disgro program 19 . The residues were then protonated using Leap in Amber14 suite of programs 20 . The contentious cases, especially for histidines were resolved by computing their pKa with the H++ program 21 . Six histidines were protonated (residues 245, 310, 332, 419, 431 and 489), each one yields a +1 charge. In order to achieve a neutral charge for the system, the two SO4− groups from the buffers have been removed and a Na+ cation was added to the model (see Figure S1).

Then, the 5’-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA) substrate was replaced by an intermediate of the bioluminescence reaction (Int). It is composed with an AMP fragment and the luciferin moiety. The charge of the Int residue is -3, one minus charge is due to phenolate part of the luciferin, another one is due to the phosphate part of the AMP, and finally the last one corresponds to the deprotonation of the carbon C1 from the thiazolone group of the luciferin. This last proton has been added on the histidine His 245, thus becoming Hip.

The AMBER99ff force field was used to model the residues of the protein. The Int was described using parameters developed by our group 10,22,23 while the atomic charges of the 5 ACS Paragon Plus Environment


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Int residue come from preliminary QM/MM calculations. A dioxygen molecule has been added into the previous file at a distance of 8.5 Å to the carbon C1 of the Int substrate. Both oxygen atoms from O2 have a charge equal to 0, and a bond distance of 1.21 Å. The resulting model is named 4G36-Int-O2 . Classical dynamics simulations were made with Amber14 program 20 . The model was solvated with TIP3P water molecules within a cube box, ensuring a solvent shell of at least 15 Å around the solute. The resulting system contained roughly 28000 water molecules and 90000 atoms in total. The system was heat from 100K to 300K in 20 ps. Then, under NPT conditions with T=300 K and P=1 atm a 5 ns dynamic using periodic boundary conditions was realized with a 2 fs time step. During these simulations pressure and temperature were maintained using the Berendsen algorithm 24 with a coupling constant of 5 ps and SHAKE constraint were applied to all bonds involving hydrogen atoms 25 .

The approach of O2 inside the protein has been computed using an umbrella sampling simulation 26 . A set of MD simulations are carried out, in each of which a harmonic ”umbrella” potential is applied to keep the bond constrained to a desired value. In details, the umbrella sampling has been realized using a constraint called Dumbrella corresponding to the distance between the two centers of mass of the fragments C1-C2 and O1-O2.

distance(M C(C) M C(O) ) = Dumbrella

(1)

with MC(C) the center of mass of the fragment C1-C2 and MC(O) the center of mass of the fragment O1-O2. The simulation is conducted constraining the values of Dumbrella from 8.5 Å to 1.5 Å, with a step of 0.2 Å. For each step an equilibration simulation of 50 ps was followed by a 150 ps 6 ACS Paragon Plus Environment

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production simulation. For each step the lowest energy conformation from the production part has been collected. This yields a set of structures along the path followed by the O2 to reach the Int substrate. To generate a free energy profile along the approach path, the bias introduced by the umbrella potential was removed using the weighted histogram analysis method (WHAM) 27,28 .

The quantum mechanics/molecular mechanics (QM/MM) calculations were performed using a coupling scheme 29 between Molcas 30 and Tinker (Molcas 8.0/Tinker). The electrostatic potential fitted (ESPF) method 29 was used to compute the interaction between the Mulliken charges of the QM subsystem and the external electrostatic potential of the MM fragments within 9 Å from the QM subsystem. The microiterations technique 31 was used to converge the MM subsystem geometry for every QM minimization step. The Int substrate was divided into 3 parts (see Figure 3). The central part containing the thiazolone cycle and the phosphate group corresponds to the QM subsystem of the molecule, i.e. the region that will be described at the QM level of theory. The rest of the molecule i.e. the benzothiazole cycle and the adenosine moiety were considered as MM subsystem. It would have been better to include the benzothiazole cycle as well as the π conjugated orbitals in the QM part. However, it is far too consuming in computational resources. Besides, in several analogues of the oxyluciferin, even if no benzothiazole cycle is present the bioluminescence is still achieved in good proportion 1,32 . The two interfaces between the QM and the MM part of the molecule Int were modelled with the link-atom scheme (LA) 33 . The two cuts were done on first a Csp3 − Csp3 bond between the phosphate group and the ribofuranose part and on a Csp2 − Csp2 bond between the benzothiazole and the thiazolone cycle. No issues have been observed during the QM/MM calculations concerning the choice of the frontier between



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the QM and the MM parts in the Int substrate. Finally the O2 molecule was included in the QM subsystem. The charge of the QM subsystem is equal to -2.

To compute the approach of O2 two constrains are used, defined as distance(C2 − O2) + distance(C1 − O1) = Dcoord 2

(2)

distance(C2 − O4) = Dester

(3)

We performed MS-CASPT2/MM single point calculations 34 with the ANO-RCC-VTZP basis set 35,36 on the SA-CASSCF/MM optimized geometries. A 10 electrons in 8 orbitals (10-in-8) active space was chosen for the SA-CASSCF/MM calculations 37,38 together with 3 states for both singlet and triplet states and with a level-shift of 0.1 39 . In details, the active space corresponds to 8 electrons in 6 orbitals for the QM subsystem localised on the Int moiety. It represents the π conjugated systems localised on the thiazolone cycle and the C=O bond. We have considered 3 π orbitals and 3 π ∗ orbitals, centred on the atoms C1, C2, O3, N1, C4 and the lone pair on S1. For the O2 we have chosen 2 electrons in 2 orbitals corresponding to two π ∗ orbitals of the molecular oxygen. This active space is quite small especially for the O2 molecule, but is a good compromise regarding computational limitation and calculation length. The presence of the lone pair on the sulphur in the active space is pointless but no 10-in-8 active spaces have converged without this lone pair. Its presence might be related to the fact that this orbital also interacts with others π orbitals of the thiazolone moiety. Despite its limited size, the active space chosen contains the orbitals involved in the formation of the dioxetanone. The different orbitals used are listed in Figure


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S2 to S8. The optimized geometries of the models listed in the publications are available in the Table S1 to S9. The atom numbering is explained in figure S9.

Figure 3: Graphical representation of the two substrates: Int and O2 . The different subsystems QM and MM are drawn on the figure.

Results The four first subsections describe the approach of the oxygen molecule and the departure of AMP. This is then discussed in terms of finding a reasonable pathway towards the formation of the cyclic peroxide structure.

The approach of the O2 molecule through classical molecular dynamics In order to fully understand how the O2 molecule binds to the Int substrate we have decided to compute the whole approach leading to formation of the dioxetanone ring, and thus start from the O2 molecule entering the luciferase. Branchini and co-workers have discussed the conformation of the protein during the reaction 18 and described a funnel between the bulk and the cavity of the luciferase as a possible entrance for O2 . We have decided to put the dioxygen molecule at the entrance of this funnel at a distance of 8.5 Å to the carbon C1 of 9 ACS Paragon Plus Environment


the Int substrate (see Figure S10). An umbrella sampling MD simulation using the constraint Dumbrella between the two centers of masses of the bonds C1-C2 and O1-O2 show that the O2 can move inside the protein without any energetic barrier. From Dumbrella = 8.5 Å to 3.5 Å there are no major changes to the global energetic profile. However, from constraint 3.5 Å to 1.5 Å, the constraint strength is not effective enough to move the dioxygen closer to Int. The dioxygen stays in the same conformation and at the same distance from the substrate Int. The value Dumbrella = 3.5 Å seems to be the limit at which quantum interactions have to be taken into account. The free energy profile graph is shown in Figure S11.

Figure 4: Graphical representation for the model Int-full, i.e. after the umbrella sampling simulation and a MM minimization. The Int and O2 substrates are represented by a licorice model while the residue Hip 245 and the water molecule 675 are represented by a sphere model. Then a MM minimization is done on the structure obtained with the constraint Dumbrella fixed at 3.5 Å without any constraint. The O2 stays at about Dumbrella = 3.5 Å and displays several electrostatic interactions with the protein environment. For example, we can see the presence of a H-bond network between O2 , the residue Hip 245 - the one protonated with the hydrogen from the substrate Int - and a water molecule (water n˚675). The bond distance C1-O1 is 3.84 Å and the distance C2-O2 is 3.16 Å, which indicates that the bond C2-O2 10 ACS Paragon Plus Environment

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should be easier to form than the C1-O1. This structure is used as the starting point for QM/MM calculations and is named Int-full (see Figure 4).

The coordination of the O2 molecule with QM/MM calculations

Figure 5: Representation of the steps used in the formation of the dioxetanone ring with MS-CASPT2//CASSCF/MM starting from the Int-full model. In order to compute the coordination of the dioxygen molecule to the Int leading to the formation of the dioxetanone ring, bonds breaking and forming have to be described and we 11 ACS Paragon Plus Environment


therefore used MS-CASPT2//SA-CASSCF/MM calculations. Two different pathways have been chosen for this study. The first one follows the direct approach of O2 to the Int substrate inside the protein and corresponds to structures named Int-full. For the second one, we first compute the breaking of the bond between the luciferin and the AMP moiety before the approach of O2 , the structures are thus named Int-cut. This second approach represents the formation of the dioxetanone ring after the departure of the AMP. Two different constraints are used, one called Dcoord and one called Dester . The constraint Dcoord corresponds to the movement of the O2 molecule towards the Int substrate while the Dester corresponds to the breaking of the ester bond between the luciferin and the AMP. The constraint Dester is only used in the subsection "Approach through the departure of AMP". For each pathway, the geometry optimization was performed in the singlet state or in the triplet state. When the name of the structure contains the letter "s", it represents a structure optimized in the singlet pathway, for the letter "t" it corresponds to the triplet pathway. The four different computational procedures to obtain the pathways are sum up in the Figure 5. Figures 6, 7 and 8 represent respectively the energy graphics and two snapshots for a direct approach and in the Figures 9, 10 and 11 the same for the approach on the Dester constraint. Main structural parameters are listed in Table S10.

Direct approach We have computed the energetic pathway from Dcoord = 3.5 Å to Dcoord = 1.5 Å starting from the structure Int-full described in Figure 4. From 3.5 to 2.3 Å the energy of the different states, i.e. S0 , S1 , T1 and T2 are nearly degenerate. The energy increases slowly to reach a value of about 0.4 eV for the model Int-s-full and Int-t-full for Dcoord = 2.3 Å when


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Figure 6: Energies in eV of the direct approach when reducing the constraint Dcoord . The point at 0 eV corresponds at the energy of the structure Int-full. In red square, the energetic pathway for S0 ; in orange square for S1 ; in green triangle for T1 and in blue triangle for T2 . compared to the starting energy of Int-full with Dcoord = 3.5 Å. During this approach, some of the orbitals centred on Int mix with the orbitals of O2 (see Figure S2 and S3). From the observation of the orbitals, it is difficult to predict which bond will be created first, i.e. C1-O1 or C2-O2, the distance C1-O1 is 2.4 Å and C2-O2 is 2.2 Å (see Figure S12).

From Dcoord = 2.2 Å to 1.5 Å we observed the splitting of the pathway followed by singlet states and the one followed by triplet states.

For the singlet states, the structure optimized at Dcoord = 2.2 Å shows the formation of the bond between C1 and O1 (see Figure 7). The formation of the bond leads to an



Figure 7: Graphical representation for the model Int-s-full (2.2) i.e. with Dcoord = 2.2 Å. The Int and O2 substrates are represented by a licorice model while the residue Hip 245 and the water molecule n˚675 are represented by a sphere model. The dashed lines represent the distances C1-O1 and C2-O2. important loss of energy, with a calculated value of -1.48 eV in comparison with the starting point. No transition states have converged during the formation of the bond, in consequence the activation barrier estimated is 0.4 eV, the energy computed from the previous point D ˙ coord = 2.3 ÅThe creation of the bond between the Int substrate and the O2 molecule has a large impact on the geometry of the system. First, the Int substrate loses its planarity, the improper dihedral angle C4-N1-C2-C1 takes a value of 30˚ in Dcoord = 2.2 Å. Thus the C1 atom becomes closer to the O1 atom of O2 leading to the formation of a C1-O1 bond of 1.5 Å. As a consequence, the O2 atom is push away, and the distance C2-O2 increases to 2.9 Å. Secondly, the shape of the orbitals of the active space changes when the bond C1-O1 is created (see Figure S4). The sulphur-centred orbital disappears, substituted by several orbitals showing the σ and σ∗ nature of the bond C1-O1. One interesting point concerns the orbitals centred on the dioxygen. Both orbitals now show an interaction with the lone pair of the nitrogen N1 of the Int substrate, the nitrogen lone pair may have quite an impact of the efficiency on the bioluminescence reaction. However, at the end of the approach, i.e. Dcoord = 1.5 Å the formation of the second bond C2-O2 is not observed. 14 ACS Paragon Plus Environment

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Figure 8: Graphical representation for the model Int-t-full (1.8) i.e. with Dcoord = 1.8 Å. The Int and O2 substrates are represented by a licorice model while the residue Hip 245 and the water molecule n˚675 are represented by a sphere model. The dashed lines represent the distances C1-O1 and C2-O2.

If we follow the triplet states, the energy keeps rising from Dcoord = 2.2 Å to Dcoord = 1.5 Å and reaches a value of 2.27 eV (62 kcal/mol) compared to the energy of Int-full with Dcoord = 3.5 Å. The formation of a bond between C2 and O2 of 1.43 Å is observed at Dcoord = 1.8 Å (see Figure 8). During the triplet states pathway, the activation barrier estimated is ˙ 3.12 eV, which corresponds to the values of the point D coord = 1.6 ÅAgain, no transitions states have been obtained. In term of geometry, the improper dihedral angle C4-N1-C2-C1 loses its planarity and its value equals 29˚. In term of the shape of the orbitals, the O2 centred orbitals induce the formation of a σ bond between C2 and O2 (see Figure S5). For the Dcoord = 1.5 Å the second bond between C1 and O1 is formed. The resulting dioxetanone ring is obtained with C1-O1 = 1.4 Å and C2-O2 = 1.6 Å. Unfortunately, if we remove the constraint and minimized the structure Int-t-full with Dcoord = 1.5 Å the dioxetanone ring breaks, and the resulting structure only show the presence of the bond C1-O1 (1.43 Å) while the bond C2-O2 is 2.50 Å.



The direct approach does not allow the formation of the dioxetanone ring, thus a new pathway has to be designed to form the expected final product.

Approach through the departure of AMP

Figure 9: Energies in eV of the approach through the departure of AMP. The point at 0 eV corresponds to the structure Int-full. In red square, the energetic pathway for S0 ; in orange square for S1 ; in green triangle for T1 and in blue triangle for T2 . In order to have the formation of the peroxide ring and thus the bioluminescence, the AMP moiety has to break away from the Int substrate. The splitting can happen at three different steps of the mechanism. The AMP can leave after the formation of the dioxetanone ring, resulting in a addition-elimination process. However, in the previous approach, the calculations do not show the formation of the dioxetanone ring, neither the spontaneous breaking of the ester bond leading to the departure of the AMP moiety. Thus we have to force the AMP to break away by using a new constraint Dester . We can use this constraint before the formation of the second bond between Int and O2 , resulting in a nucleophilic substitution SN 2 if the dioxetanone is formed when the AMP is leaving or a SN 1 if the AMP leaves first, followed by the formation of the bond. This approach is studied on the structure obtained at Dcoord = 1.5 Å. The constraint Dester can also be used on the structure Int-full 16 ACS Paragon Plus Environment

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with Dcoord = 2.3 Å, before the formation of the first bond C-O (see Figure 9).

Figure 10: Graphical representation for the model Int-s/t-cut. The structure has been obtained after the constraint Dester = 1.8 Å on the structure Int-s/t-full (2.3). The Int and O2 substrates are represented by a licorice model while the residue Hip 245 and the water molecule n˚675 are represented by a sphere model. The dashed lines represent the distances C1-O1 and C2-O2 used in the Dcoord constraint and the distance C2-O4 used in the Dester constraint. The approach starting from the structure optimized at Dcoord = 2.3 Å will be discussed below while the one with the structure optimized at Dcoord = 1.5 Å is described in the SI (Figure S13 and S14).

Starting from the structure Int-full with Dcoord = 2.3 Å, we applied the constraint Dester = 1.8 Å to yield the optimized structures Int-s-cut and Int-t-cut for the singlet states and triplet states, respectively. Both the Int-s-cut and Int-t-cut structures show the same geometry characteristics. The distance C1-O1 is equal to 3.42 Å while C2-O2 is equal to 2.88 Å. The O2 substrate creates a hydrogen network with both Hip 245 and the water molecule n˚675 (see Figure 10). The MS-CASPT2/MM calculated single point displays an energy of +0.71 eV for Int-s-cut and +0.82 eV for Int-t-cut. The departure of AMP also lifts the energy degeneracy between singlet and triplet states as observed in the direct approach before the formation of the bond C1-O1. The breaking of the bond C2-O4 is done by a heterolytic 17 ACS Paragon Plus Environment


cleavage, all the electronic density can be observed on the centred orbital of the oxygen atom O4 of the AMP moiety. Then we applied the constraint Dcoord = 1.5 Å on both singlet and triplet structures. In contrast with the direct approach, no intermediate structures are computed along the path of the coordination of O2 . We have also checked that the bond C2-O4 does not reunite during the approach of O2 even if the Dester constraint is not anymore applied. . We first focus on the singlet approach. During the optimization, the bond C1-O1 forms first, followed by the bond C2-O2, leading to the formation of the dioxetanone ring. A final minimization calculation gives the Int-s-cut-dioxetanone structure (see Figure 11). In this structure the bond distances and the angles are consistent with previous calculations of dioxetanone intermediates 2,9 , all the data can be find in Table 1. The energy of the minimized structure lies at -1.51 eV, and the S0 -S1 gap reaches 5.31 eV. In term of orbitals the one centred of the sulphur disappears while we observed the presence of the orbitals σ and σ ∗ in the active space for the bond C1-O1 but not for the bond C2-O2 (see Figure S7).

Figure 11: Graphical representation for the model Int-s-cut-dioxetanone. The structure has been obtained after a minimization of the structure Int-s-cut (1.5), where Dcoord = 1.5 Å. The Int and O2 substrates are represented by a licorice model while the residue Hip 245 and the water molecule n˚675 are represented by a sphere model. The dashed lines represent the distances C1-O1 and C2-O2 used in the Dcoord constraint and the distance C2-O4 used in the Dester constraint.


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When following the triplet approach the formation of a dioxetanone ring is obtained. As observed in the direct approach, the bond C2-O2 is first created followed by the C1-O1 one. A final minimization gives rise to the Int-t-cut-dioxetanone structure (see Figure 12). The energy of the minimized structure lies at +1.51 eV, and the T1 -T2 gap reaches 2.05 eV. In term of orbitals the one centred of the sulfur is still present unlike in Int-s-cut-dioxetanone. The σ and σ ∗ orbitals of the C1-O1 bond are also present in the active space but there are no orbitals describing the bond C2-O2 (see Figure S8). For both Int-s-cut-dioxetanone and

Figure 12: Graphical representation for the model Int-t-cut-dioxetanone. The structure has been obtained after a minimization of the structure Int-t-cut (1.5), where Dcoord = 1.5 Å. The Int and O2 substrates are represented by the licorice model while the residue Hip 245 and the water molecule n˚675 are represented by the sphere model. The dashed lines represent the distances C1-O1 and C2-O2 used in the Dcoord constraint and the distance C2-O4 used in the Dester constraint. Int-t-cut-dioxetanone structures, the formation of the dioxetanone ring influences the nearby environment. When we look at the surrounding, the hydrogen network formed between Hip 245, the water molecule n˚675 and the O2 molecule has disappeared. However, the water molecule n˚675 is still close to the Int substrate and is in interaction with the Hip 245 and an oxygen atom of the phosphate group of the AMP (see Figures 11 and 12).



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Table 1: Selected important geometric parameters for structures with a dioxetanone ring and comparison with reference SA-CASSCF results obtained in vacuo 2,9 . Bond lengths are in angstroms and angles are in degree.

C1-O1 C2-O2 C1-C2 O1-O2 C1-O1-O2-C2

Int-t-full with Dcoord (1.5) 1.40 Å 1.60 Å 1.55 Å 1.41 Å -25˚

Int-s-cutdioxetanone 1.49 Å 1.36 Å 1.50 Å 1.40 Å -3˚

Int-t-cutdioxetanone 1.50 Å 1.37 Å 1.50 Å 1.41 Å -1˚

Dioxetanone geometry ref 2,9 1.49 Å 1.33 Å 1.53 Å 1.57 Å 3˚

Discussion The O2 moving inside the protein Looking at the model 4G36-Int-O2 , the one before the umbrella sampling dynamic, we can see that the proton transferred from Int to Hip 245 is in interaction with the Int substrate with a distance of 2.02 Å between the carbon C1 and the hydrogen (see Figure S15 and Table S11). The two molecules are indeed close to each other, which shows that the residue 245 is able to deprotonate the Int substrate as supposed in the first part of the mechanism. Then, after the umbrella sampling simulation, the dioxygen is well stabilised inside the cavity. In details, the oxygen O2 is involved in a H-bond network with a hydrogen of Hip 245, a water molecule and the phosphate part of the Int substrate. A proton transfer may be possible between the Hip 245 and the O2 molecule, resulting in a new possible mechanism. If the proton bonds the dioxygen, a hydroperoxide species is obtained like in one of the hypotheses discussed in the introduction (see Figure S16). However it has not been tested in this publication.

As stated before, the O2 molecule is free to move inside the protein and can come close to the Int substrate. However when we removed the constraint Dumbrella applied between O2 20 ACS Paragon Plus Environment

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and Int, the dioxygen is moving away and most of the time going out of the protein through two different channels. These two channels correspond to the one used by O2 during the umbrella sampling and the one supposed to be used by the ATP and the D-luciferin when entering the active site in the bioluminescent mechanism. Moreover, it seems impossible for the dioxygen to coordinate from the other side of the Int substrate as several residues obstructed the pathway.

The formation of the dioxetanone ring During the umbrella sampling simulation, the deprotonation of the hydrogen on C1 yield a -1 charge delocalised around the thiazolone cycle (and a total -3 charge for the Int substrate), which is in accordance with the charges of the force field used for the substrate. However, the active space chosen for the structure Int-full, initially with the π conjugated system of Int, the lone pair centered on S1 and two lone pairs of O2, shows that the π orbital of C1 has a 1 electron occupation and the sum of the electrons on the 2 actives orbitals of O2 is 3 electrons. Thus the active space chosen shows the spontaneous formation of radicals on both Int and O2 at the beginning of the QM/MM simulation, i.e. when O2 is at 3.5 Å from the intermediate. The presence of these radicals is observed until the formation of the first bond, C1-O1 or C2-O2. Unfortunately it is quite difficult to know when the electron transfer leading to the formation of the radicals is done during the approach before the QM/MM calculation as the classical MD does not give such information. Spin-orbit calculations have been performed ˙ computed singlet-triplet spin-orbit coupling for distance Dcoord between 3.5 Å and 2.9 ÅThe is too low to observed singlet to triplet interconversion. But as the spin-orbit coupling keep increasing when the distance Dcoord increases, the intersystem crossing is likely to happen when O2 is further from Int than in the the first QM/MM structure optimized. The presence



of these radicals favors a mechanism involving a dioxygen superoxide. This kind of mechanism is not new and has been observed in bioluminescent proteins or mono-oxygenase and dioxygenase protein and referenced in literature 40–42 . In fireflies, the presence of radicals in the mechanism has also been investigated and proved experimentally 16 .

The present study gives a description of the main steps of the formation of the dioxetanone ring. In Figure 5, the energetic pathway in function of the constraint distance Dcoord is drawn. From Dcoord = 3.5 Å to 2.3 Å, the energy of the electronic states S0 , S1 , T1 and T2 are nearly degenerate. In details, the energy of S0 is slightly lower than T1 (about 0.05 eV) except for Dcoord = 3.1 Å where the difference is 0.16 eV. The state degeneracy can favor the intersystem crossing between the singlet and the triplet state. Indeed, the O2 enters the protein in a triplet state while the dioxetanone intermediate is known to lay in a singlet state to achieve the bioluminescence emission. The structures Int-s/t-cut obtained when applying the constraint Dester on Int-full with Dcoord = 2.3 Å lift the state degeneracy but the difference of energy between S0 and T1 is small, about 0.09 eV. The departure of the AMP moiety does not impact much the stability of the rest of the system. This can be explain because there are no orbital of the AMP moiety taken into account in the active space, even though the phosphate group is include in the QM part. The use of the constraint Dester is necessary if we want to drive away the AMP moiety. Indeed, as we do not include in the active space the σ orbital of the bond C2-O4, it prevents the spontaneous breaking and departure of the AMP moiety.

Then when the Dcoord constraint decreases, the singlet manifold and the triplet manifold split into two different mechanisms. For the singlet pathway, the bond C1-O1 is created first


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at Dcoord = 2.2 Å followed by the C2-O2 bond. The formation of the C1-O1 bond stabilizes the energy of the system of about 1.5 eV. However it is not possible to form the dioxetanone ring by following the direct approach. The possible reason can be that the AMP moiety blocks the geometry and forbids the formation of the dioxetanone ring. Indeed, when the AMP moiety is first driven away, using the Dester constraint, the dioxetanone ring can be obtained. The energy of Int-s-cut-dioxetanone, the structure with the dioxetanone ring is lower in energy than the starting point by -1.51 eV (-35 kcal/mol). For the triplet pathway, the first bond formed is C2-O2 at Dcoord = 1.8 Å. Like the singlet manifold, the formation of the dioxetanone ring is possible only when the AMP moiety is pushed away from the Int substrate. However, the values of the energy calculated are higher when compared to the starting structure, and to the singlet manifold. For example, the energy of Int-t-cutdioxetanone is + 1.7 eV in comparison with Int-full with Dcoord = 3.5 Å. It is thus difficult to explain how the formation of the dioxetanone ring is possible following the triplet states, because the least amount of energy needed is 39 kcal/mol. This tends to favor the singlet manifold as a pathway to form the dioxetanone ring.

Conclusion In this publication we have investigated the approach of the dioxygen molecule inside a proteinic environment in order to form the dioxetanone ring. This reaction corresponds to an important step in the bioluminescence mechanism in fireflies and has never been studied in details before. The theoretical approach combines classical molecular dynamics, using an umbrella sampling simulation and hybrid QM/MM calculations with the hypothesis of the deprotonated intermediate by a near Histidine residue. We can, with our results, propose the mechanism described in Figure 13 as a possible mechanism to explain the formation of 23 ACS Paragon Plus Environment


the dioxetanone ring by a superoxide ion in the firefly bioluminescence reaction involving first the deprotonation of the Int susbtrate by His 245. First, the dioxygen molecule can move freely inside the protein, with no energetic barriers observed along the umbrella sampling simulation. When coming closed to the Int substrate, O2 is well stabilised by the nearby environment, i.e. water molecules and residues. This is only possible when assuming the deprotonation of the Int substrate by a nearby base, here the histidine 245. Thus, we also notice that the proton transfer between Int and the histidine 245 may be possible due to their proximity. Secondly, QM/MM calculations with a MS-CASPT2//SA-CASSCF/MM approach has been realized to form the dioxetanone ring. One important result obtained is the presence of radicals species in the mechanism, with the dioxygen recovering one electron from the Int substrate. Indeed, when O2 approaches the deprotonated Int substrate, the electronic density shows a spontaneous electron transfer from the Int to the O2 , leading to the formation of a superoxide ion before the formation of the dioxetanone ring. Then, depending of the pathway, singlet or triplet, the mechanism shows some differences. For the singlet manifold, the bond C1-O1 is formed first, while for the triplet manifold the bond C2-O2 formation is observed first. Moreover, when following the singlet pathway, the last structure obtained shows a decrease of the energy of about 1.5 eV, whereas for the triplet pathway the energy has increased of about 1.8 eV. Thus, the formation of the dioxetanone ring is favored with singlet states. Third, in order to have the formation of the dioxetanone ring, the AMP moiety of the Int substrate has to be driven away. In our approach, the departure of the AMP fragment is the only way to form the dioxetanone ring for both the singlet and triplet states. In this study we describe a chemical reaction embedded in the protein environment. The


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interactions between the two substrates and the environment are essential to get insights on the mechanism steps and cannot be put aside. The better comprehension of the bioluminescence mechanism in fireflies can be used to increase the understanding of others bioluminescent species where few details are nowadays known.

Figure 13: Proposed mechanism of firefly bioluminescence, the number near structures corresponds to respective paragraph in conclusion.

Supplementary Information Graphical representation of the position of protonated histidines and Na+ , CASSCF orbitals of the active space used in structures described in this publication, Cartesian coordinates of stationary points, Graphical representation of the position of O2 in the funnel of the luciferase before MD, PMF profile for the umbrella sampling simulation, Structural parameters of the dioxetanone cycle during the O2 approach, Graphical representation of the model Intfull (2.3), Representation of the steps used in the Int-1.5-cut approach and step by step explanation, Graphical representation of the structure Int-t-1.5-cut-dioxetanone, Graphical



representation of the structure 4G36-Int-O2 , Comparison of the distance between C1 and the newly proton added on Hip245, Graphical representation of the possible equilibrium between the superoxide and hydroperoxide ion. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements R.B.P. and I.N. would like to acknowledge support from the ANR Biolum project (ANR16-CE29-0013). R. L. acknowledges support by the Swedish Research Council (grant 201603398) for financial support.

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MM Study of the Formation of the Dioxetanone ... - ACS Publications

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