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Electrostatic Catalysis Induced by Luciferases in the Decomposition of the Firefly Dioxetanone and Its Analogs Jian-Ge Zhou, Shan Yang, and Zhen-Yan Deng J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b08000 • Publication Date (Web): 23 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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The Journal of Physical Chemistry

Electrostatic Catalysis Induced by Luciferases in the Decomposition of the Firefly Dioxetanone and its Analogs Jian-Ge Zhou,*,+ Shan Yang,+ Zhen-Yan Deng‡ +

Department of Physics, Atmospheric Science, and Geoscience, Jackson State University, Jackson, Mississippi 39217, United States ‡

Department of Physics, Shanghai University, Shanghai 200444, China

ABSTRACT: The variations of the barrier heights in the decomposition of the firefly dioxetanone and its analogs with the electrostatic field produced by the active site amino acid residues in the firefly luciferase are examined by a DFT study for the high energy intermediates of the three luciferins. The positive electric field along the long-axis direction of the luciferins lowers the activation energy and acts as an electrostatic catalyst in the thermolysis process. The calculated barrier heights for the firefly dioxetanone and its analogs surrounded by the firefly Photinus pyralis luciferase show that the energy barrier of the firefly dioxetanone is lowered by the luciferase, but is raised for the other analog. Thus the thermolysis rate is enhanced for the natural D-luciferin and reduced for the other by the firefly luciferase, which elucidates why the luciferase acts as a catalyst for the natural D-luciferin but makes some luciferins emit weaker light signals.

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INTRODUCTION Firefly bioluminescence has been an attractive subject in the scientific community for decades due to its broad applications in gene expression, cell trafficking networks, enzyme activity, disease progression, and environmental control.1-3 Bioluminescence imaging is one of the most popular methods for visualizing biological processes in vitro, in live cells, and even in whole organisms.2,3 The bioluminescent reaction involves D-luciferin (substrate), adenosine triphosphate (ATP), oxygen molecule, magnesium cation and firefly luciferase (enzyme).2 The light emitter, the oxyluciferin in the luciferase active site, is produced via the decomposition of a high energy intermediate – firefly dioxetanone (DO) formed from the D-luciferin(-1)-AMP.4-13 One of the goals in bioluminescence research is to find a way to modulate the color and intensity of the emitted light. The light color depends on not only the makeup of the luciferin molecule but also the luciferase which surrounds the luciferin14,15. In bioluminescence, the mutant luciferase can modulate the light color of the luciferin15-19 by changing the local electrostatic field (LEF) produced by the mutant luciferase. As the bioluminescence imaging tool is applied to examine tissues, the absorption and scattering of light by tissues results in strong fading of bioluminescent signals. Therefore, one needs light emitters that can radiate red or near-infrared light with high intensity,20 which can penetrate tissues more easily than other colors. The bioluminescence technology for multicomponent imaging needs more light-emitting luciferins to provide different colors of bioluminescent light. Recently, several heterocyclic analogs of the luciferins have been synthesized, and these modified heteroaromatic luciferins can be easily uptaken by cells and have similar biodistributions as the natural D-luciferin (LH2) in vivo.20-22 The heterocyclic analogs emit different colors with various light intensities. For example, recent studies observed significant and different changes on bioluminescent intensities from modified lucifereins, which


2

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are obtained by replacing the benzothiazole ring of the LH2 with the benzoxazole (BoLH2) or benzothiophene (BtLH2).21,22 The oxidized structures of modified LH2 are compared with that of LH2 in Figs. 1a-1c. Although extensive research work has been conducted on tuning the light color,14-32 it is still not clear what factors mainly affect the light intensity and why some emitters emanate more brightly than others. Since the decomposition of the DO is a rate-determining step in the firefly bioluminescence,4-13 the examination of the thermolysis process might reveal the mechanism of light intensity modulation.

On the other hand, electrostatic interactions play a critical role in enzyme catalysis, where enzymes employ the protein architecture to apply electrostatic fields to their bound substrates.33,34 The firefly luciferin, located in the pocket of the firefly luciferase, is surrounded by the charged side-chains of amino acid residues (Fig. S1).28-30 The LEF exerted by the active site is correlated with the enzyme’s catalytic rate enhancement.33,34 Firefly bioluminescence consists of two step reactions (adenylation and oxidation) of firefly luciferin, the ATP, O2 and Mg2+ catalyzed by firefly luciferase. The luciferyl-adenylate and pyrophosphate are first produced, then the excited-state of the oxidized luciferin (oxyluciferin) is generated, and its subsequent relaxation to the ground state releases photons of yellow-green light (Fig. 2).2 In the oxidation process, the firefly dioxetanone is formed, and its thermolysis produces CO2 in ground state and part of the oxyluciferin in the first excited singlet state (see the 3rd row of Fig. 2).4-13 The thermolysis of the firefly dioxetanone in the anionic form - DO (Fig. 1d) is carried out through an asynchronous two-stage pathway with one transition state and the double crossing of the potential energy surfaces of the ground state (S0) and the first excited singlet state (S1).11-13 These two conical intersection spots are needed for efficient firefly bioluminescence. The


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decomposition of the firefly dioxetanone in the neutral form - neutral DO (Fig. 1g) requires a two-step pathway with two transition states connected by the intermediate state, in which the S0, S1 and the first excited triplet state (T1) are S +y N

a) +x

b)

O

nearly degenerate in the biradical region and

N

lead to nonadiabatic transitions. This entropic

N

S

N

S trap

c)

S

S

N

N

C

e)

d)

N

N

g)

N O2

N

h)

S

emission

is

directly

The ΦBI is the yield of the production of a O2

S

N

O1 N S

compound.11-13 The light intensity of the

and bioluminescent quantum yield (ΦBI).35,36

S

S

excited-state carbonyl

N

O1 N

an

proportional to both the catalytic reaction rate

S

O2 f)

to

bioluminescent

S

S

S

O2 O1

leads

O1 N

photon from a single reactant, which includes 1) the efficiency of the chemical reaction; 2)

S

Figure 1. The oxidized structures of the three the yield of crossing to the excited state; 3) the luciferins. a) LH2, b) BoLH2, c) BtLH2. The structures of the DO for d) the ground state, fluorescent yield of the excited oxidized e) the transition state and f) product. The 4-13 The relative light intensity is structures of the neutral DO for g) the ground luciferin. state, h) the transition state. directly proportional to · ΦBI,35,36 where . is the energy difference between two activation energies of the DO and its analogs. If the is sizable, the catalytic rate constant is a primary factor controlling the relative light intensity. To search for new emitters with higher light intensity, one needs to examine what catalytic role of electrostatics plays in the thermolysis of the DO as well as its analogs BoDO and BtDO (the high energy intermediate for BoLH2 and BtLH2, see Fig. S2). The relation between the energy barriers of the thermolysis transition states and the LEF can display how the


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LEF lowers the activation energies and enhances the catalytic rate. This correlation can tell what emitters emanate stronger light signals and which amino acid residues in the firefly Photinus pyralis luciferase (PpyLuc) should be substituted to increase the light intensity, thus it provides the criterion on choosing the optimal pair of the modified luciferin and corresponding mutant luciferase.37,38 The PpyLuc is a catalyst in the firefly bioluminescent process,1-3 then one may ask if the PpyLuc lowers barrier height of the DO, in other words, if the DO in the environment of the PpyLuc possesses a lower activation energy than that of the DO in a gas phase. However, theoretical study that investigates how the PpyLuc (or its induced LEF) affects the energy barrier in the thermolysis process remains absent. The computational comparison of the energy barriers among various modified luciferins in a gas phase and in the PpyLuc environment can explain why some emitters emanate more brightly than others.

In this contribution, the geometries of the ground and thermolysis transition states of the DO, BoDO and BtDO in the presence of the LEF induced by the active site amino acid residues are optimized via the density functional theory.39-41 The geometric changes of the ground and transition states under the LEF along x- (the long-axis direction of the molecules, see Fig. 1a), yand z-direction are characterized by the O-O bond length between the O1 and O2 (Fig. 1e). The O-O bond length variation under the LEF is discussed from the covalent bond strength (ground state), diradical interaction (transition state), electrostatic interaction (LEF) and the dipole moments of the states. To study the electrostatic catalysis of the luciferase, the variations of the barrier heights with the LEF are examined for the DO, BoDO and BtDO. The energies of the ground and transition states under the LEF are evaluated to check which state plays main contribution to the modification of the activation energies. The response of the dipole moment x-


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component to the LEF along the x-direction in the ground and transition state is discussed to interpret the origin of why the ground state plays an important role in the variation of the barrier heights. The results show that the positive LEF created by the PpyLuc along x-direction lowers the activation energy and acts as an electrostatic catalyst. To further investigate how the PpyLuc affects the thermolysis of the high energy intermediates of the three luciferins, the activation

Figure 2. Adenylation reaction forming luciferyl-adenylate (1st row). Oxidation reaction forming oxyluciferin excited state (2nd and 3rd rows). energies for the transition states of the DO, BoDO and BtDO in the pocket of the PpyLuc (Fig. 1e and Fig. S3) are examined by using the ONIOM method.42 The energy barriers of the DO, BoDO and BtDO surrounded by the PpyLuc show that the PpyLuc lowers the activation energy for the DO but raises the barrier height for the BoDO. As a result, the thermolysis rate is enhanced for the DO and weakened for the BoDO by the PpyLuc, which elucidates why the PpyLuc acts as a catalyst for the LH2 and the BoLH2 emits the weakest light among the LH2, BoLH2 and BtLH2.

COMPUTATIONAL METHOD


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The density functional theory (DFT) at the CAM-B3LYP/6-311+G(d,p) level was applied to optimize the geometries of the ground and transition states for the DO, BoDO and BtDO in the presence of the LEF. Three unrestricted open-shell density functionals UCAM-B3LYP39, UM062X40 and UWB97XD41 with the long-range correction have been tested with 6-311+G(d,p) basis set, and they have similar trends (Fig. S4). The results of the UCAM-B3LYP functional best fit the experimental relative light intensities for the DO, BoDO and BtDO.20-22 The close-shell approximation is appropriate for the optimization of the ground state geometry, but poor for describing open-shell biradical system. If the close-shell method with an amount of HF exchange is applied to calculate the transition state, the result is unreliable due to the instability of the Kohn-Sham wave function.43,44 The unrestricted open-shell CAM-B3LYP functional is appropriate for the optimization of the biradical system (transition state). The α and β spatial symmetry of the initial guess orbital of singlet open-shell system before the self-consistent field iteration should be broken, which is the key of symmetry broken technology.13 The transition states have been fully optimized, and the force constants were determined analytically in the analysis of harmonic vibrational frequencies for all the complexes. When the DO, BoDO and BtDO is in the pocket of the PpyLuc (Fig. S1), the starting geometries come from the structure of the AMP and the PpyLuc, in which the PpyLuc is simulated by the twenty active site residues.31 The coordinates of the twenty active site residues and AMP were selected from the PDB structure 4G37, where the firefly luciferase is in the second catalytic conformation.45,46 These active residues are Arg218, His245, Gly246, Phe247, Thr251, Leu286, Ala313, Ser314, Gly315, Gly316, Arg337, Gln338, Gly339, Gly341, Leu342, Thr343, Thr346, Ser347, Ala348 and Lys443. The geometries of the DO, BoDO and BtDO surrounded by active molecules were optimized by using the ONIOM approach,42 where the DO, BoDO or BtDO was in the higher


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layer, while the remaining molecules were in the lower layer. The geometry of the DO, BoDO or BtDO was optimized by the DFT at the UCAM-B3LYP/6-311+G(d,p) level, and the active site molecules were treated by the UFF force field with an electrostatic embedding scheme.47 The UCAM-B3LYP/6-311+G(d,p)/UFF calculations were implemented in the implicit solvent via the conductor-like polarizable continuum model.48 The implicit solvated simulation in the dibutyl ether with the dielectric constant 3.05 was performed to account for the average effect of the protein and water molecules.31,32 The Gaussian 09 package of programs49 was used for the computation.

RESULTS AND DISCUSSION The oxidized structure of the LH2, the OxyLH2, has the keto and enol form,50-65 and the firefly dioxetanone can take the anionic form denoted by DO which carries one negative electric charge (Fig. 1d and Fig. 2) or neutral form denoted by neutral DO (Fig. 1g and Fig. 1h).66,67 We first investigate the DO with the thiazoline and benzothiazole ring linked by the C2−C2′ bond (Fig. 2), then compare it with the neutral DO. The oxidized BoLH2 or BtLH2 is obtained by replacing the benzothiazole ring by the benzoxazole (OxyBoLH2) or benzothiophene (Oxy BtLH2),21,22 and their high energy intermediates BoDO and BtDO are built on from their corresponding anionic form. The averaged LEF induced by the PpyLuc around the luciferins is in the range of [−150

Å

, 150

Å

] along the x-, y- and z-direction.28-30 The structures of the ground and

transition states for the DO, BoDO and BtDO in the gas phase without the LEF were optimized. The geometries of the high energy intermediate ground and transition states are characterized by the O-O bond length between the O1 and O2 (Figs. 1d-1e, Figs. S2-S3). For the ground states, The O-O bond lengths are 1.47Å for the three high energy intermediates. In the thermolysis


8

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process of the DO, BoDO and BtDO, the transition states are treated as the biradical states, in which one applies the symmetry-broken approach to get more accurate transition structures.4,11-13

a)

c)

b)

d)

Figure 3. a) The variation of the O-O bond length (Å) between the O1 and O2 for the transition state of the DO with the LEF, the abscissa axis Ei represents Ex or Ey (mV/Å). b) The variation of the energies (au) of the ground and transition state for the DO with the Ex. c) The dependence of the x-component of the dipole moments (Debye) of the ground and transition state on the Ex. d) The comparison between the activation energy ETS and E - Δµx·Ex. When the ground states evolve to the transition states, the O-O bond lengths prolong from the original 1.47Å to 1.75Å, 1.74Å and 1.75Å for the DO, BoDO and BtDO (Fig. 1e, Fig. S3). The calculated barrier heights for the DO, BoDO and BtDO in the gas phase without the LEF are 14.00, 12.79 and 13.58 kcal/mol respectively, and they are at the same order. The computational


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energy barriers are consistent with the experimental values of the dioxetane derivatives, which are between 12 kcal/mol and 24 kcal/mol.68 Now we examined the structures of the ground and transition states for the DO, BoDO and BtDO in the gas phase with the x-, y- and z-directional LEF (Fig. 1a). The O-O bond lengths of the ground states for the three compounds with the LEF along x-, y- and z-direction are around 1.47 Å. In the ground states, the O-O bond is formed covalently and the bond strength is much stronger than the electrostatic interaction as the LEF is in the range of [-150

Å

, 150

Å

], thus the

LEF changes the O-O bond length very little. When there is no LEF, the O-O bond length stretches from 1.47 Å (ground state) to 1.75 Å (transition states). For the transition states, the OO bond lengths with the LEF along x- and z-direction are around 1.75 Å. When the LEF in the yaxis increases from -150

Å

to 150

Å

(Fig. 3a), the O-O bond length for the DO decreases from

the 1.85 Å to 1.65 Å, and for the other two compounds the O-O bond lengths have similar variation range. As the LEF along the y-direction increases from -150

Å

to 150

Å

, the Ey

induces a larger change in the y-component of the dipole moment (Δµy=4.13 Debye) than the changes in µx (Δµx =2.12 Debye) and µz (Δµz=2.71 Debye) that LEFs along the x-, z-direction cause respectively. This observation indicates that the Ey changes the charge distribution and modifies the O-O bond length more easily than the LEF along other two directions in the transition states (Fig. S5).

The dependence of the energy barriers ETS on the LEF is investigated for the DO, BoDO and BtDO and is displayed in Table 1. The energy barriers decrease when the electric field along xand y-direction increases from -150

Å

to 150

Å

, but they increase when the LEF along z-


10

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direction increases. The variation of the activation energy is most sensitive along x-direction, and the positive Ex lowers the activation energy. For instance, the ETS varies from 25.6 kcal/mol (Ex=-150

Å

) to 5.3 kcal/mol (Ex=150

Å

) for the DO, which indicates the LEF along the

positive x-direction acting as an electrostatic catalyst. Three energy barrier differences are 20.3, 19.3 and 19.2 kcal/mol for the DO, BoDO and BtDO when the Ex varies from -150 150

Å

Å

to

. Among three high energy intermediates, the DO is most sensitive to the electric field in x-direction. To lower the thermolysis energy barrier of the high energy intermediates by mutagenesis, the above relation between the ETS and the LEF suggests that one should substitute the active site amino acid residues so that the resulting mutant luciferases produce less negative

LEF along the long-axis direction of the luciferin. Figure 4. The variations of the energy barriers with the Ex for the DO and neutral When one searches for new luciferins emitting DO. high intensity of light, one should look for the luciferins whose high energy intermediates are very sensitive to the LEF along the x-axis. The mutant luciferase producing positive LEF in the x-direction paired with the luciferins that their high energy intermediates have very sensitive ETS might be the potential optimal pairs with strong light intensity.37,38

To check how the energy barriers are modulated by the LEF, the energies of the ground and transition state of the DO in the presence of the LEF along x-direction (Ex) are evaluated and illustrated in Fig. 3b. As the Ex varies from -150

Å

to 150

Å

, the energy of the ground state

increases explicitly from -1628.5550 au to -1628.5231 au; but the energy of the transition state


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varies between -1628.5147 au and -1628.5139 au (Fig. 3b). For the three high energy intermediates, the energy barriers are modified by mainly changing the energy of the ground state. To get a hint why the variation of the ground state energy is considerably greater than that of the transition state, we examined the electric charges on the O1 (QO1) and O2 (QO2) atom. When Ex = -150

Å

, for the ground state the QO1 and QO2 are -0.296 and -0.253 (QO2 - QO1 =

0.043), and for the transition state the QO1 and QO2 are -0.351 and -0.346 (QO2 - QO1 = 0.005), which indicates that the electric charge of the transition structure is spread over the molecule more evenly than that of the ground state (Table S1). At Ex = 150

Å

, the charge differences

between the O2 and O1 (QO2 - QO1) are 0.059 and 0.017 for the ground and transition state, which suggests that the dipole moment of the transition state should be less than that of the ground state. The variation of the dipole moments of the ground and transition state with the LEF along the x-direction was calculated and displayed in Fig. 3c, which shows that the x-component of the dipole moment of the ground state is greater than that of the transition state. In the presence of the electric field, the energies of the ground and transition state can be approximated as: E(G) ≈ E0(G) - G · and E(TS) ≈ E0(TS) - TS · , where E0(G) and E0(TS) are the energies of the ground and transition state without the electric field, TS and G are the dipole moments of the transition and ground state, is the electric field, and E(G) and E(TS) are the energies of the ground and transition state with the LEF. For simplicity, suppose the LEF is only along xdirection, the energies of the ground and transition state are reduced to E0(G) - µx(G)·Ex and E0(TS) - µx(TS)·Ex, where the µx(G) and µx(TS) are the x-components of the dipole moment for the ground and transition state. Since the corresponding µx(G) is greater than the µx(TS) (Fig. 3c), the variation of the ground state energy with the Ex is considerably greater than that of the transition state (Fig. 3b). The activation energy ETS = E(TS) - E(G) under the LEF becomes E -


12

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Δ · with Δ = TS - G. Here we should mention that the TS and G consist of two parts, the first one is the permanent dipole moment which is determined by the molecular structures; the second one is related to the electric field and the polarizability tensor of the molecules.29 When the LEF is along x-direction, the calculated activation energy ETS agrees well with the E -

Δµx·Ex, as illustrated in Fig. 3d. Thus, the electric field modifies the energy barrier through its ), and it lowers the activation energy when coupling with the dipole moment difference (Δ · the LEF along certain directions (see Table 1).

Table 1 The energy barriers (kcal/mol) of the three transition states of the DO, BoDO and BtDO under the local electrostatic field (mV/ Å) along x-, y-, and z-direction. electric field

Ex E

E

Ey E

E

E

Ez E

E

E

E

-150

25.6

23.8

25.2

16.3

16.9

17.4

12.2

10.7

11.1

-100

21.3

19.9

21.1

15.5

15.2

16.1

12.6

11.0

11.6

-50

17.5

16.2

17.1

14.7

14.1

15.3

13.1

11.7

12.4

0

14.0

12.8

13.6

14.0

12.8

13.6

14.0

12.8

13.6

50

10.7

9.7

10.3

13.2

11.6

11.9

15.1

14.3

15.2

100

7.8

7.0

7.3

12.5

10.4

11.0

16.5

16.1

17.3

150

5.3

4.5

6.0

11.7

9.0

9.7

18.1

18.2

19.6

Now let us examine how the barrier height of the transition state for the neutral DO changes with the LEF. Since the variation of the activation energy is most sensitive along x-direction for the DO in which the O6’ carries a negative charge, we study the response of the barrier height of the neutral DO to the Ex. In the decomposition of the firefly dioxetanone, the DO has only one


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transition state TSO-O which is obtained by stretching the O1-O2 bond, but the neutral DO has two transition states: the first one is the TSO-O corresponding to the O1-O2 bond stretch, and the second one is the TSC-C corresponding to the further C-C bond stretch.13 To check how the energy barriers of the DO and neutral DO respond differently to the Ex, we compare their energy barriers for the transition states TSO-O, and the results are illustrated in Fig. 4. For the DO, when the Ex increases from -150

Å

to 150

Å

, the energy barrier decreases from 25.6 kcal/mol to 5.3

kcal/mol (Fig. 4), which indicates that the LEF along the positive x-direction acts as an efficient electrostatic catalyst. For the neutral DO, the energy barrier increases from 20.9 kcal/mol to 27.4 kcal/mol, which shows that barrier height of the DO is more sensitive to the LEF than that of the neutral DO.

To see how the PpyLuc affects the thermolysis of the high energy intermediates of the three luciferins, the activation energies for the transition states of the DO, BoDO and BtDO surrounded by the PpyLuc and AMP are evaluated via the ONIOM method.42 The starting geometry of the PpyLuc simulated by the twenty active site amino acid residues and the AMP is obtained from the PDB structure 4G37, which is the firefly luciferase in the second catalytic conformation.31 The C-terminal domain of the luciferase in the first adenylation conformation undergoes a ̴140° rotation to adopt the second oxidative conformation.45,46 These active site residues include Arg218, His245, Gly246, Phe247, Thr251, Leu286, Ala313, Ser314, Gly315, Gly316, Arg337, Gln338, Gly339, Gly341, Leu342, Thr343, Thr346, Ser347, Ala348 and Lys443.31 Our calculated energy barriers of the DO, BoDO and BtDO in gas phase (without the PpyLuc, AMP or LEF) are 14.0, 12.8 and 13.6 kcal/mol. The energy barriers of the DO, BoDO and BtDO surrounded by the PpyLuc and AMP are calculated by the ONIOM approach,42 and


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they are 6.2, 29.6 and 13.2 kcal/mol respectively. To account for the bulk effect of the PpyLuc and buried water molecules, the barrier heights of the DO and its analogs surrounded by the PpyLuc and AMP in the implicit solvent (dibutyl ether) were calculated by using the conductor-

His245

Phe247

2.73

2.68

3.03 2.64

2.93

Ser347

2.60

Arg218 Lys443 Phosphate (AMP) Figure 5. The selected amino acid residues in the PpyLuc and AMP surrounding the DO, the minimal distances between them and the ground state structure of the DO are displayed.

like polarizable continuum model.48 The calculated energy barriers of the DO, BoDO and BtDO complexed with the active site molecules in this implicit solvent are 7.6, 31.2 and 14.1 kcal/mol respectively. The energy barrier differences between the model of the twenty proximal active amino acid residues and that of the twenty close-by active amino acid residues in the dibutyl ether solvent with the dielectric constant 3.05 for the DO, BoDO and BtDO are 1.2, 1.6 and 0.9


15


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kcal/mol respectively. The slight changes of the energy barriers indicate that to model the protein as the twenty proximal active amino acid residues in the dibutyl ether solvent with the dielectric constant 3.05 is appropriate for calculating the energy barrier shift caused by the protein64,65. By comparing the activation energies between the gas phase DO, BoDO and BtDO and the ones complexed with the active site molecules in the implicit solvent, we find the energy barrier of the DO is lowered by 6.4 kcal/mol, but the energy barrier of the BoDO is raised by 18.4 kcal/mol, which shows that the PpyLuc acts as a catalyst for the D-luciferin, and the BoLH2 luciferin emits the weakest light among the three substrates.20-22 On the other hand, the calculated energy barrier of the transition state TSO-O for the neutral DO in gas phase is 23.8 kcal/mol. When the neutral DO is complexed with the active site molecules in the implicit solvent, the corresponding energy barrier is 23.1 kcal/mol, so the PpyLuc modifies its energy barrier slightly.

The peak of the light intensity curve occurs at the beginning of the bioluminescent process.15-22 The initial light intensity is directly proportional to both the catalytic reaction rate and bioluminescent quantum yield,35,36 Since the initial substrate concentration is much greater than the Michaelis constant Km, the catalytic reaction rate becomes directly proportional to both the catalytic rate constant (kcat) and the initial concentration of the enzyme.20-22 The initial concentration of the enzyme is the same for the natural luciferin and its analogs, and the wavelengths of the LH2, BtLH2 and BoLH2 are around 500 nm, then their relative light intensity (Irel) is directly proportional to the product of the catalytic rate constant and quantum yield, i.e., Irel = kcat·ΦBI. As observed in Refs. 20-22, the measured relative quantum yields for the LH2 and BtLH2 are 100% and 70%,20-22 and the experimental catalytic rate constants are 1.9 x 10-1 s-1 (LH2) and 6.7 x 10-3 s-1 (BtLH2). The measured relative light intensities of the flash are 100%


16

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(LH2) and 4.9% (BtLH2).20-22 The calculated relative light intensities via I = kcat·ΦBI are 100% (LH2) and 2.5% (BtLH2), and match the experimental values. Although the relative quantum yield of the BtLH2 is 0.7 times of that of the LH2, the relative light intensity of the BtLH2 is only 4.9% of that of the LH2, which indicates that the relative light intensity is directly proportional to the product of the catalytic rate constant and quantum yield, and that the catalytic rate constant plays a dominant role in the relative light intensity in this experimental case. Since the activation energy (ETS) is the highest energy along the potential energy curve of the ground state in the decomposition of the DO or its analogs,5,6 the catalytic rate constant can be approximated to

. Thus the relative light intensity is directly proportional to

· ΦBI, where is the

energy difference between two activation energies of the DO and its analogs. If the is sizable, the catalytic rate constant is a primary factor controlling the relative light intensity. The calculated energy barriers of the DO, BtDO and BoDO complexed with the active site molecules in the implicit solvent are 7.6, 14.1 and 31.2 kcal/mol, thus the order of the relative light intensities should be DO > BtDO > BoDO, which is in line with the experimental relative light intensities: 100% (LH2), 4.9% (BtLH2) and 1.0% (BoLH2).21,22

Since the DO, BoDO and BtDO have distinct constituents, they interact with the active site amino acid residues in the PpyLuc differently. To characterize how the PpyLuc interacts with the substrates, we selected some active site amino acid residues (Arg218, His245, Phe247, Ser347, Lys443) and the AMP to display the minimal distances between them and the ground state DO (Fig. 5). To manifest the different interactions between the high energy intermediates and the PpyLuc, Table S2 lists the minimal distance between the selected residues (including the AMP) and the structures (the ground and transition state) of the DO and its analogs. The high


17


Page 18 of 33

energy intermediates of the luciferins bind to the multiple charged active site residues in a precisely oriented way. These active site residues and the AMP have different orientation and distances with respect to the three substrates, thus they produce different electric field around the DO, BoDO and BtDO, which interprets why the DO, BoDO and BtDO have different activation energies in the PpyLuc and AMP. To see how different orientation and distances in the ground and transition state of the DO, BoDO and BtDO create different electric field on the molecules, the LEFs produced by the PpyLuc and AMP at the position of the O1 and O2 atom of the ground state DO(G), BoDO(G), and BtDO(G) as well as the transition state DO(T), BoDO(T) and BtDO(T) were calculated and listed in Table 2. The results showed that the electric fields are different among three compounds. For the same substrate, the electric fields are also different for the ground and transition state. Such a difference in the produced electric field results in the different energy barriers.

Table 2 The LEF (Å) produced by the PpyLuc and AMP at the O1 and O2 atom of the ground state DO(G), BoDO(G), and BtDO(G) as well as the transition state DO(T), BoDO(T) and BtDO(T). O1

O2

Ex

Ey

Ez

Ex

Ey

Ez

DO(G)

27.68

-26.02

9.60

-2.05

-0.39

3.18

BoDO(G)

0.59

-0.89

-0.95

0.46

-0.18

-0.49

BtDO(G)

21.44

-11.78

-8.06

6.15

0.95

-3.75

DO(T)

28.20

-18.30

-2.80

3.35

0.53

-0.14

BoDO(T)

-0.49

-0.54

-1.59

0.02

-0.87

0.16

BtDO(T)

13.86

-13.92

0.76

3.41

0.88

-0.98


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CONCLUSION The geometries of the ground and thermolysis transition states of the DO, BoDO and BtDO in the presence of the LEF induced by the active site amino acid residues have been examined by the DFT method17 with unrestricted open-shell functional UCAM-B3LYP in the 6-311+G(d,p) basis set. For the ground states, the O1-O2 atoms bond together covalently, and the bond strength is much stronger than the electrostatic interaction in the range of [-150

Å

, 150

Å

], so the LEF

keeps the O-O bond length at almost the same value. For the transition states, the O-O bond lengths under the LEF along x- and z-direction change slightly. When the LEF in the y-axis increases, the O-O bond length for the DO decreases, which has been interpreted as the Ey causes a larger change in the dipole moment µy than that the Ex and Ez induce in µx and µz. The variations of the energy barriers with the LEF have been studied for the DO, BoDO and BtDO, and it has been found that the barrier heights decrease when the electric field along x- and ydirection increases, but they increase when the LEF along z-direction increases. The change of the activation energy is most sensitive along x-direction, and the positive Ex lowers the activation energy considerably which indicates that the positive LEF created by the PpyLuc along xdirection is an electrostatic catalyst. The evaluated energies of the ground and transition states under the LEF have revealed that the ground state plays main contribution to the modification of the barrier heights, which can be attributed to the fact that the transition states have weaker dipole moments than those of the ground states. The energy barriers of the DO, BoDO and BtDO surrounded by the PpyLuc and AMP in the implicit solvent have demonstrated that the PpyLuc lowers the activation energy for the DO but raises the barrier height for the BoDO. As a result, the thermolysis rate is enhanced for the DO and reduced for the BoDO by the PpyLuc,


19


Page 20 of 33

which elucidates why the PpyLuc acts as a catalyst for the LH2 and the BoLH2 luciferin emits the weakest light among the LH2, BoLH2 and BtLH2. Since the Lys443 plays the significant role in the oxidation process,15 it might be interest to lift the Lys443 to the high layer and investigate how it or its substitutes affects the barrier height. The luciferase possesses two conformations: adenylation and oxidation,45,46 in the bioluminescent process we plan to explore the energy barriers in the two luciferase conformations in order to justify the existence of the second oxidative structure from the thermolysis perspective.

Supporting Information The ground and transition state structures of the DO, BoDO and BtDO; the transition energy barriers of the DO in the gas phase calculated by the three functionals UCAM-B3LYP, UM062X and UWB97XD; the dipole moments of the transition state for the DO with the LEF along x-, y- and z-direction; the electric charges on the O1 and O2 atom (QO1 and QO2) and their charge difference (QO2 - QO1) when the LEF along the x-direction changes; the minimal distance between the selected amino acid residues in the PpyLuc and the ground state structures (G) (transition state structures (T)) of the DO, BoDO and BtDO; the coordinates of the ground and transition states of the DO, BoDO and BtDO in the PpyLuc and AMP.

AUTHOR INFORMATION Corresponding Authors *E-mail:[email protected]. Phone: (601) 979-3758


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ACKNOWLEDGEMENTS This work is supported by the National Institutes of Health (NIH) under Award Number SC2DE027240, the Innovation Program of Shanghai Municipal Education Commission (Grant 13ZZ079), and the “085 project” of Shanghai Municipal Education Commission.

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in

DMSO/TMG

and

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bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety, J. Org. Chem. 2011, 76, 902908.


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Electrostatic Catalysis Induced by Luciferases in ... - ACS Publications

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