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. 2024 Apr 9;4(4):1591–1604. doi: 10.1021/jacsau.4c00075

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

Tai-Ping Zhou †, Jianqiang Feng †, Yongchao Wang †, Shengying Li ‡,*, Binju Wang †,*
PMCID: PMC11040706  PMID: 38665654

Abstract

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P450 NascB catalyzes the coupling of cyclo-(l-tryptophan-l-proline) (1) to generate (−)-naseseazine C (2) through intramolecular C–N bond formation and intermolecular C–C coupling. A thorough understanding of its catalytic mechanism is crucial for the engineering or design of P450-catalyzed C–N dimerization reactions. By employing MD simulations, QM/MM calculations, and enhanced sampling, we assessed various mechanisms from recent works. Our study demonstrates that the most favorable pathway entails the transfer of a hydrogen atom from N7–H to Cpd I. Subsequently, there is a conformational change in the substrate radical, shifting it from the Re-face to the Si-face of N7 in Substrate 1. The Si-face conformation of Substrate 1 is stabilized by the protein environment and the π–π stacking interaction between the indole ring and heme porphyrin. The subsequent intermolecular C3–C6′ bond formation between Substrate 1 radical and Substrate 2 occurs via a radical attack mechanism. The conformational switch of the Substrate 1 radical not only lowers the barrier of the intermolecular C3–C6′ bond formation but also yields the correct stereoselectivity observed in experiments. In addition, we evaluated the reactivity of the ferric-superoxide species, showing it is not reactive enough to initiate the hydrogen atom abstraction from the indole NH group of the substrate. Our simulation provides a comprehensive mechanistic insight into how the P450 enzyme precisely controls both the intramolecular C–N cyclization and intermolecular C–C coupling. The current findings align with the available experimental data, emphasizing the pivotal role of substrate dynamics in governing P450 catalysis.

Keywords: P450, diketopiperazine, enzyme mechanisms, QM/MM, molecular dynamics, dimerization reaction, C−N bond formation

1. Introduction

Small molecule natural products (NPs) can be valuable resources for treating a variety of diseases and disorders.1−4 NPs consist of a large variety of compounds, including polyketides (PKs), nonribosomal peptides (NRPs), terpenoids, and alkaloids.5−9 Among these, the tryptophan-linked dimeric diketopiperazine (DKP) derivatives possess distinctive structural architecture and extensive bioactive properties, such as anticancer, antitumor, antiviral, and neuroprotective activities.10−14 Cytochrome P450s (CYPs),15−23 a superfamily of heme-dependent enzymes, are demonstrated to be crucial for the biosynthesis of DKPs.7,8,24−26 Recently, two homologous P450 enzymes (Scheme 1), NascB and NznB, have been characterized to catalyze the dimerization of cyclo-(l-tryptophan-l-proline) (1) to generate (−)-naseseazine C (2) and (+)-naseseazine B (3), respectively.27−30 These types of transformations involve both the intramolecular C–N coupling and intermolecular C–C coupling, which are unique for the catalysis of P450s.31−34

Scheme 1. Dimerization Reactions Catalyzed by NascB and NznB.

Scheme 1

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In addition to NascB and NznB, another homologous enzyme named NasF5053 has been characterized by Qu and co-workers.28 NasF5053 demonstrated high catalytic activity toward (1). Of note, the double mutant S284A-V288A predominantly produced compound (2). The high-resolution (1.68 Å) crystal structure of this mutant in complex with the native substrate (Figure 1) showed that the active site is occupied by two units of (1), with each occupying a distinct pocket. In addition, Substrate 1 is anchored by an array of H-bonding networks extended from residues K289, while Substrate 2 is stabilized by G286 and E314.

Figure 1.

Figure 1

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Crystal structure of NasF5053 (PDB ID: 6VZB) in complex with two units of (1), and an enlarged view of the active site is displayed in the right box. Key hydrogen bonds are indicated by dashed lines. The distances are given in Å.

Scheme 2 summarizes two plausible mechanisms that emerge from experimental and computational investigations.13,27,28 Based on structural information and the preliminary simulations, the catalytic route A has been proposed in NasF5053 by Qu and co-workers.28 In this route, the reaction is initiated by the hydrogen atom transfer (HAT) from the N1–H of Substrate 1 to Cpd I, the well-recognized active species in P450 catalysis.35−40 This HAT step would afford an N1-centered indolyl radical intermediate (4), which may undergo further protonation to form a cation radical intermediate (5). This is followed by intramolecular C2–N7 coupling via a Mannich-type reaction, which could be coupled to the proton release from N7, leading to a pyrroloindoline radical intermediate (7) for the subsequent intermolecular C–C coupling reaction. Such a mechanism could be inferred from the structural information, indicating that the indole N1–H group of Substrate 1 is in the proximity of the heme-Fe, making it an ideal candidate for the HAT reaction (Figure 1). Furthermore, density functional theory (DFT) calculations indicated that the N1-centered radical resulting from HAT at N1 is significantly more stable than the N7-centered radical formed through HAT at the N7 site.13,27 However, recent molecular dynamics (MD) investigations by Sherman and Houk showed that Substrate 1 may experience a conformational movement, leaving its N7–H close to Cpd I.29 Additionally, the mechanism of the proton shuttle in route A remains elusive, as there are no apparent residues within the crystal structure that could mediate this process.34 Therefore, there is still a lack of direct evidence supporting pathway A.

Scheme 2. Two Possible Dimerization Mechanisms in P450 NascB.

Scheme 2

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Sherman and co-workers have proposed mechanism B in NzeB based on DFT model calculations.27 In this pathway, the reaction initiates with HAT from the N7–H group of Substrate 1, resulting in the formation of the diketopiperazyl radical (6). Unlike route A, intramolecular C2–N7 bond coupling within (6) may bypass the complicated proton shuttle. For the C2–N7 coupling, it was proposed that the N7 radical may attack the Si-face of the C2=C3 double bond, resulting in the correct configuration at the C2 site. This is followed by the intermolecular C3–C6′ bond formation via a radical mechanism. The subsequent rearomatization via the HAT from (8) intermediate to the ferry-hydroxo species (Cpd II) affords the final product (2). Interestingly, the crystal structure of NzeB (PDB ID: 6XAI) has been characterized, which bears quite similar active site architecture and substrate binding characteristics as those of NasF5053. Notably, the N1 of Substrate 1 is close to the heme-Fe (with a distance of 2.96 Å), while the N7 is 6.90 Å away from heme-Fe (Figure S1), indicating a preference for hydrogen atom abstraction (HAA) from the N1–H over the N7–H. However, the following MD study by the same group showed that the Substrate 1 repositioning may bring the N7–H group closer to the Cpd I species, thereby facilitating the generation of (6).29

Despite the aforementioned mechanistic insights, the molecular mechanism of the P450-catalyzed biosynthesis of DKPs is not fully elucidated, especially regarding the protein environment effects in dictating the regio- and stereoselective C–N and C–C coupling. Extensive studies have shown that the protein environment is key to the activity and selectivity of enzyme catalysis, while the neglect of such an effect in the QM model calculations may give biased descriptions of the kinetic and thermodynamic properties of enzymatic processes.37,41−52 In this study, we reexamined the molecular mechanism of the P450 NascB with the combined MD simulations53,54 and quantum mechanical/molecular mechanical (QM/MM) calculations.55−59 Particularly, we systematically explored all conceivable catalytic pathways originating from two distinct binding modes of Substrate 1. Our multiscale calculations reveal that pathway B, which involves a conformational movement of the substrate radical, is the most favorable reaction pathway. Such a conformational switch of the substrate radical not only reduces the barrier of the intramolecular C–N bond coupling but also aligns with the correct stereoselectivity as observed in experiments.

2. Methods

We used MD simulations for substrate binding conformational sampling. The hybrid QM/MM technique was employed to determine the catalytic mechanism. The umbrella sampling simulation was utilized to investigate the process of the conformational transformation. The details of these steps will be discussed in the following section, with the QM calculation method available in the Supporting Information.

2.1. System Setup and Classical MD Simulations

The initial structure coordinates of the enzyme–substrate complex were taken from the Protein Data Bank (PDB ID: 6VZB).28 The missing residues were completed using the Modeller program.60 In the previously suggested catalytic mechanism, the reaction is triggered by Cpd I species. The O atom above heme-iron was modeled as the oxo atom of Cpd I.61,62 The protonation states of all titratable residues were assigned according to their pKa values calculated by the PROPKA procedure.63,64 Overall, all Glu and Asp residues were deprotonated, while the Arg and Lys residues were protonated. His109, His166, His177, His182, His205, and His 336 were protonated at the epsilon nitrogen, while His254, His262, His264, His316, His344, and His 381 were protonated at the delta nitrogen. The histidine residues His9, His188, and His194 were doubly protonated at both the epsilon and delta nitrogen atoms. The protonation states and local hydrogen-bonded networks were visually checked by using the VMD program.65 The leap tools of the Amber package66 were used to give the coordinates of the missing hydrogen atoms. For further MD simulations, the general Amber force field (GAFF)67 was utilized for both substrates, whereas the partial atomic charges were received from the RESP method68 at the B3LYP-D3BJ/6-31G(d,p)69,70 level of theory. The AMBER ff14SB force field71 was employed for the protein residues. The parameters of Cpd I were taken from a previous study,72 while the force field parameters of Cpd II were prepared with the “MCPB.py” tool73,74 of AmberTools18.66 The parmchk2 tool from AmberTools18 was used to generate the missing parameters of the substrate. Several counter (sodium) ions were added to the protein surface with the leap tools to neutralize the total charge of the system. The resulting system was solvated in a rectangular box filled with the TIP3P75 model waters extending up to a minimum distance of 16 Å from the enzyme’s surface.

Using the above-prepared system, we started a consecutive two-step minimization of the targeted system to avoid steric clashes that occur during the system setup. In the first step, the added water molecules were subjected to minimization, while in the second step, the whole system was minimized using 10 000 steps of steepest descent and 10 000 steps of conjugate gradient algorithm, respectively. Then, the system was gently heated from 0 to 300 K under an NVT ensemble for 300 ps with a weak restraint of 25 kcal/mol/Å. To gain a uniform density after the heating process, 1.0 ns of density equilibration was performed under the NPT ensemble at a target temperature of 300 K and with a target pressure of 1.0 atm. The temperature value was kept using the Langevin thermostat76 and the pressure was maintained with the Berendsen barostat77 with a collision frequency of 2 ps and pressure relaxation time of 1 ps. This was followed by further equilibrations for 10 ns under an NPT ensemble without restraints for each system. Finally, three independent 200 ns productive MD simulations under the NPT ensemble were performed. The SHAKE method78 was used to constrain the hydrogen bond while particle mesh Ewald and appropriate cutoff distances (∼10 Å) were used to treat the long-range electrostatic and van der Waals forces, respectively. All MD simulations were carried out with the GPU version of the AMBER 18 package.66 The CPPTRAJ module of Amber 18 was used to analyze all of the results. The VMD software package was used for visualizing the MD simulation results.

2.2. QM/MM Calculations

All QM/MM calculations55,56 were carried out using representative snapshots extracted from the MD simulation of the enzyme–substrate complexes. The representative snapshots were chosen based on the near-attack conformation from the MD trajectory (i.e., the shortest distance between the oxo of Cpd I and the target N–H). The ChemShell software package57,79 was employed to perform all QM/MM calculations. This is done in combination with TURBOMOLE80 for treating the QM zone and DL_POLY81,82 for the MM region with the AMBER force field. The electronic embedding scheme83 was implemented to incorporate the polarizing effect of the enzyme environment on the QM region. Hydrogen link atoms with the charge-shift model were applied to treat the QM/MM boundary. The QM/MM system contains all proteins, counterions, and solvation waters within 12 Å of the protein. A truncated heme-porphyrin ring and the proximal cysteine as −SCH3 were incorporated in the QM zone (see Figure S2 for the QM zone). The active region during QM/MM calculations includes all of the residues and solvent molecules within a distance of 16 Å from the Fe center. During QM/MM geometry optimization, the QM region was treated with the hybrid UB3LYP density functional,69 which was demonstrated to be practical for the study of P450 systems.37,45,84−88 For geometry optimization and scanning calculations, the def2-SVP basis set89 (labeled B1) was used. Then, the single-point calculations were conducted with the higher basis set def2-TZVP (labeled B2). The initial transition state (TS) structures were taken from the highest point of the potential energy surface (PES). Then, the TS was fully optimized using the dimer optimizer90 implemented in the DL-FIND code.91 The natural bond orbital (NBO) spin population80 was obtained at the UB3LYP/B2 level. The Grimme DFT-D3BJ corrections70 were added to all QM/MM calculations.92−94 As both our calculations (refer to Tables S2 and S3) and previous studies show that S = 1/2 (doublet) and S = 3/2 (quartet) states exhibit generally similar reactivities for the Cpd I-mediated reactions,37,95−98 our discussions are limited to the doublet state in the main text. The calculated spin populations of key atoms of the involved species are shown in Table S1.

2.3. Umbrella Sampling

The umbrella sampling99 was applied to investigate the “Re → Si” conformational switch of the Substrate 1 radical species. The C2–C3–C4–C5 dihedral angle was chosen as the collective variable (CV) for the “Re → Si” transformation, which varies from −90.6 to 110.4, with a 3° interval for two adjacent windows. For each window, a 10 ns MD simulation with a basing harmonic potential (with a force constant of 200 kcal/mol/Å) was carried out to ensure sufficient overlap between two adjacent windows. During all restraint MD simulations, the covalent bonds containing hydrogen were constrained using SHAKE, and an integration step of 1 fs was used. In the production runs, a dump frequency of 1 ps–1 of the value of CV was used to collect the data needed for the potential of mean force (PMF) analysis. PMF was constructed with the weighted histogram analysis method (WHAM),100,101 using the CV data in the last 5 ns restraint production simulations of each window. Convergence was analyzed by comparing the resultant PMF with the one obtained from the CV data of the last 10 ns of restraint production. To verify the stability of the Si conformation obtained from the umbrella sampling, the structure from the umbrella sampling window (CV = 104.3°) was relaxed with 200 ns MD simulations without any restraints.

3. Results and Discussion

3.1. Substrate Binding Modes

To identify the binding modes of the two substrates, three separate 200 ns MD simulations were conducted (refer to Figures S3–S5). In line with the previous study, we observed two representative binding modes of Substrate 1 (Sub1) during the MD simulations.29 In the initial 70 ns MD simulations, Sub1 maintains a conformation where its indole N1–H is H-bonded to the Cpd I species (labeled as Conf-a), resembling the conformation observed in the crystal structure (Figure 2a).102 However, the binding mode of Conf-a is relatively unstable and rapidly transforms into another conformation after 70 ns (labeled as Conf-b), in which the DKP N7–H forms a hydrogen bond with iron-oxo (Figure 2b). Inspection of Figure 2a,b shows that the binding modes of Substrate 2 (Sub2) undergo minor changes, while Sub1 repositions significantly after 70 ns MD. Notably, in Conf-a, Sub1 engages in hydrogen-bonding interactions with Lys289, whereas in Conf-b, Sub1 is stabilized through hydrogen-bonding interactions with Val236. As both binding modes were observed during the MD simulations, both of them were considered for subsequent mechanistic investigation.

Figure 2.

Figure 2

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QM(UB3LYP-D3BJ/B1)/MM-optimized active site structure of Cpd I with the substrate for (a) Conf-a and (b) Conf-b. For the sake of clarity, unimportant hydrogen atoms are not shown. Key hydrogen bonds are indicated by dashed lines. The distances are given in Å.

3.2. Indole N–H Abstraction Pathway A

Initially, we explored the intricate reaction mechanism in pathway A (Scheme 2), where Sub1’s oxidation occurs via H-abstraction from its indole N1–H bond by Cpd I. Figure 3 depicts the QM/MM-calculated relative energy profile for pathway A, and the corresponding structures can be found in Figure S6. It is seen that the HAA from the indole NH group by Cpd I involves a low barrier of 13.8 kcal/mol, resulting in the intermediate Int1a, whose energy is 3.1 kcal/mol higher than that of RCa. Population analysis reveals that the radical is delocalized across the indole ring of Sub1 in Int1a, with a significant population at C3 (−0.43) and N1 (−0.22). Commencing from the Int1a intermediate, we explored four potential pathways, as outlined in Scheme 3. However, all of these pathways are kinetically inaccessible.

Figure 3.

Figure 3

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QM(UB3LYP/B2)/MM-calculated energy profile (in kcal/mol) for the H-abstraction from N1–H in pathway A, along with the schematic drawing of key intermediates.

Scheme 3. QM(UB3LYP/B2)/MM Calculated the Energy Barriers for All Possible Reaction Routes Emerging from Int1a.

Scheme 3

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Energies are given in kcal/mol relative to Int1a.

The first pathway involves the OH-rebound to the N1 site. However, its barrier is over 30.0 kcal/mol (Figure S7) and thus can be ruled out. In the second pathway, the OH-rebound to the C3 site also requires a high barrier of over 40.0 kcal/mol, mainly due to unfavorable substrate positioning (Figure S8). In the third route, FeIV–OH species in Int1a may perform HAA from the N7–H site, transforming the indolyl radical to a diradical species. However, this route requires a remarkable barrier of more than 50.0 kcal/mol (Figure S9). In the fourth route, we investigated the intermolecular C3–C6′ bond formation between the indolyl radical and Sub2, but the process experiences a barrier of over 50.0 kcal/mol according to our scanned energy profile (Figure S10). Obviously, the HAA from the substrate N1–H group would afford dead-end intermediate Int1a that cannot undergo further transformations.

Beginning with the Int1a intermediate, the protonation of the indolyl radical species, as suggested by Qu et al., yields a protonated species.28 To verify its possibility, we conducted further testing using the QM method. The pKa of indole radical species is approximately 4.8 lower than that of the experimental environment (∼7.5, Scheme S1),103 indicating that the protonation of the Sub1 radical is endothermic of 6.55 kcal/mol104 (estimated by 4.8 × 1.364 kcal/mol), making it thermodynamically unfavorable. QM calculations show that the protonated indolyl radical presents a high energy barrier in all assessed reactions (Scheme S1, Figure S11). In summary, all of our calculations suggest that pathway A in Scheme 2 is highly unfavorable and thus can be ruled out.

3.3. Diketopiperazine N–H Abstraction Pathway B

In this section, we proceed to investigate the dimerization reactions from the binding conformation of Conf-b. As shown in Figure 2b, the H7 atom of Sub1 forms a hydrogen bond with FeIV=O in Conf-b, implying that the FeIV=O species might catalyze HAA from the N7 bond. Figure 4 presents the QM/MM-calculated energy profiles initiated from HAA from the N7–H group (pathway B). The primarily QM/MM-optimized structures involved in the reactions are depicted in Figure 5. It is observed that HAA from the N7–H bond experiences an energy barrier of 19.0 kcal/mol, leading to the intermediate Int1b, which is 16.5 kcal/mol higher than RCb. The spin population analysis shows that one electron transfers from Sub1 to the porphyrin group during H-abstraction by FeIV=O species (Figures 5 and S12), leading to the Fe(IV)–OH state in the so-formed Int1b. This finding is consistent with earlier studies.13,27 Starting from Int1b, we first investigated the attack of N7 onto the C2 site of the indole ring via the Re-face direction, which may afford the pyrroloindoline radical intermediate. As depicted in Figure 4, the calculated barrier for this step is 23.4 kcal/mol relative to RCb. However, we found that C2 maintains the R configuration in the resulting Int2b. Starting from Int2b, we further investigated the intermolecular C3–C6′ coupling between the pyrroloindoline radical and the Sub2. As shown in Figure 4, the process requires a quite high barrier of 40.0 kcal/mol relative to that of RCb. Since the C2–H bond is close to the FeIV–OH species in Int2b, we also investigated the HAT from the C2–H to the FeIV–OH species, and the process requires a relatively high barrier of 25.6 kcal/mol compared to RCb (Figure S13). All of these observations indicate that the direct intramolecular C2–N7 coupling within Int1b may not be responsible for the dimerization reactions.

Figure 4.

Figure 4

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QM(UB3LYP-D3BJ/B2)/MM-calculated energy profile (in kcal/mol) for pathway B, along with a schematic drawing of key intermediates.

Figure 5.

Figure 5

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QM(UB3LYP-D3BJ/B1)/MM-obtained structures of key species along pathway B, shown along with the spin population of the key atoms. The distances are given in Å. For clarity, only relevant hydrogen atoms are shown.

Inspired by our previous study, we assume the diketopiperazyl radical may adopt the flexible conformation.45,105−107 Consequently, we have explored the rotation of the dihedral angle C2–C3–C4–C5 using the enhanced umbrella sampling.108−111 As indicated in Figure 6a, the rotation of the C2–C3–C4–C5 dihedral angle experiences a barrier of 6.3 kcal/mol, leading to another stable conformation that is 0.4 kcal/mol lower than the initial conformation. In the new conformation, the Sub1 radical is stabilized by hydrogen-bonding interactions with surrounding residues, such as Val236 and Lys289 (Figure 6b). In addition, the dihedral angle has increased to 101.0°, which may enable N7 to attack the C2 of indole via the “Si” conformation, leading to the S configuration at the C2 site in the formed pyrroloindoline radical intermediate. To further verify the stability of the Sub1 radical in its “Si” conformation, we have performed 200 ns fully relaxed MD simulations, showing that the system is quite stable, with an RMSD value of less than 1.5 Å (Figure S16a). The analysis112 of protein–ligand interaction for both “Re” and “Si” conformation revealed that the diketopiperazyl radical was stabilized by nonbonding interactions including hydrogen bond, hydrophobic interaction, and π–π stacking (Table S5). In addition, the nonbonding interaction between the Sub1 radical and the protein environment was calculated to be −53.5 kcal/mol in the “Si” conformation, which is comparable to the value of −55.1 kcal/mol in the “Re” conformation (Figure S17), supporting the idea that both conformations of the radical species can be stabilized by the protein environment. In comparison, we also investigated the conformational change of substrate 1 before the H-abstraction reaction. However, the conformational transformation of substrate 1 was found to be unfavorable thermodynamically in both binding modes (Conf-a and Conf-b; see Figure S18). Thus, the generation of the radical species via the H-abstraction facilitates the conformational transformation of Sub1.

Figure 6.

Figure 6

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(a) Calculated PMF (in kcal/mol) for the transformation of “Re” conformation to “Si” conformation. The reaction coordinate is defined as the C2–C3–C4–C5 dihedral angle. (b) The active site structure of the “Si” conformation. For clarity, unimportant hydrogen atoms are not shown. Key hydrogen bonds are indicated by dashed lines. The distances are given in Å.

Figure 7 presents the QM/MM-calculated relative energy profile for the dimerization reactions initiated from the “Si” conformation of the Sub1 radical, while Figure 8 illustrates the QM/MM-optimized structures of key intermediates and TSs. In Int1c, the spin population predominantly resides on the N7 atom (−0.69). Starting in Int1c, the N7 attacks the Si-face of the C2=C3 bond (Int1c → TS2c), which involves a barrier of 21.8 kcal/mol that forms Int2c and affords Int2c that is 2.4 kcal/mol higher in energy than RCb. In TS2c, the C2–N7 bond distance has shortened to 2.26 Å, compared to 3.19 Å in Int1c. In the nascent Int2c, the C3 site carries the most spin population (−0.62), which would facilitate the following intermolecular C3–C6′ bond coupling between the Int2c radical and Sub2 via a radical-mediated process. This process involves an energy barrier of 23.0 kcal/mol relative to RCb, leading to Int3c, which is 14.7 kcal/mol higher than RCb. In comparison, the calculated barrier of C3–C7′ bond coupling is 30.7 kcal/mol (Figure S19), which is in line with experiments in which the C3–C6′ coupling is favored over that of the C3–C7′ coupling. This is mainly attributed to the shorter distance of C3–C6′ (3.50 Å) relative to that of C3–C7′ (4.84 Å), suggesting that the substrate positioning is key to the selective C3–C6′ bond formation. Therefore, our calculations support that the C–C bond formation occurs via a radical-mediated mechanism, rather than a cationic Friedel–Crafts mechanism suggested before.34 In the final step, the HAT from C6′–H to Cpd II forms the aromatization product (Int3c → Int4c), which involves an energy barrier of 20.9 kcal/mol relative to RCb.

Figure 7.

Figure 7

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QM(UB3LYP-D3BJ/B2)/MM-calculated energy profile (kcal/mol) for pathway B after the conformational transformation of the Sub1 radical, along with the schematic drawing of the key intermediates.

Figure 8.

Figure 8

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QM(UB3LYP-D3BJ/B1)/MM-obtained structures of key species along pathway B after the conformational transformation of the Sub1 radical are shown along with the spin population of key atoms. The distances are given in Å. For clarity, only relevant hydrogen atoms are shown.

Inspection of Figures 5 and 8 shows that the intermolecular C3–C6′ bond coupling from the “Si” conformation (Int2c → TS3c in Figure 7) is remarkably favored over that from the “Re” conformation (Int2b → TS3b in Figure 4). This is primarily because Int2c has a significantly lower energy compared to Int2b. In Int2b, dihedral N1–C2–N7–C5 features an angle of −131.3°, which leads to significant ring strain in the newly formed five-membered ring. In contrast to Int2b, the ring strain is significantly released in Int2c, attributed to the dihedral angle C3–C4–N7–C7 measuring 174.4°. Moreover, the existence of intermolecular π–π stacking interactions (Figure S20) between the indole ring and porphyrin contributes to the additional stabilization of Int2c. Consequently, the rotation of the C2–C3–C4–C5 dihedral angle not only diminishes the barrier for the intermolecular C3–C6′ bond coupling between pyrroloindoline radical intermediate and Sub2 but also leads to the correct stereoselectivity for the dimerization reaction.

3.4. Why the O-Substituted Substrate Is Not Reactive for Dimerization Reaction

In an experiment conducted by Qu and co-workers, an O-substituted substrate (O-sub) analogue was tested, but no reactivity can be identified (Figure 9a).13 As the analogue of the O-sub does not undergo dimerization by NascB, it was assumed that the H-abstraction from the indole N–H could be key to the dimerization by NascB. To rationalize such experiment observation, we performed 200 ns MD simulations for the Cpd I state of P450 NascB with the O-sub. Our MD simulations demonstrate that the system can reach a stable convergence state after 60 ns, as evidenced by calculated RMSD (Figure S21a). In a snapshot from the converged trajectory, it is seen that the N7–H group of the O-sub is far away from Cpd I, with an average distance of ∼10 Å (Figure S21b). In addition, the substituted oxygen atoms of the O-sub do not form any hydrogen-bond interaction in the active center (Figure 9b). Consequently, our MD simulation results indicate that the inappropriate binding conformation of the O-substrate renders it highly unfavorable to initiate HAA from the N7–H group, potentially explaining why this substrate does not exhibit reactivity in the dimerization reaction.

Figure 9.

Figure 9

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(a) O-substituted substrate (O-sub) analogue tested in NascB. (b) Representative structure of the Cpd I with the O-sub from MD simulation. For clarity, unimportant hydrogen atoms are not shown. Key hydrogen bonds are indicated by dashed lines. The distances are given in Å.

3.5. Can Ferric-Superoxide Act as a Potential Oxidant in P450 NascB

In very recent research, the ferryl-intermediate was suggested as an oxidant to facilitate the dimerization of cyclic dipeptides by P450 AspB.113 To verify if such an intermediate could be involved in the substrate dimerization in P450 NascB, we have conducted MD simulations and QM/MM calculations on the ferric-superoxide (FeIII–OO•–)-mediated HAA from the indole N1–H of the substrate in the NascB enzyme. In line with a previous study,114 the ground state is calculated to be the open-shell singlet (OSS), as summarized in Figure 10a. In the optimized OSSRCc structure, the spin populations on the atoms of O10 and O11 are 0.54 and 0.48, respectively, while the spin population on Fe is −1.00. This is consistent with a typical OSS ferric-superoxide complex.114 Further QM/MM scanning from the OSSRCc structure shows that the energy of HAA from N1 increases steadily, with no stable intermediate that can be identified along the energy profile. Additionally, the estimated energy barrier exceeds 26.0 kcal/mol (Figure 10b). All of these findings indicate that the ferric-superoxide species lacks sufficient reactivity to initiate HAA from the substrate’s indole NH group. Such finding is consistent with the previous study by Shaik and co-workers, in which the ferric-superoxide was demonstrated to be a sluggish oxidant for the C–H hydroxylation in P450cam.115 To further compare the different reactivity between the ferric-superoxide species and Cpd I species, we computed the bond dissociation energy (BDE) of the O–H bond in different species (Figure 10c).116−119 It is seen that the BDE values of Cpd II are comparable to those of the substrate N–H bond. However, the BDE of FeIII–OOH is 21.7 kcal/mol lower than that of the Cpd II, indicating the ferric-superoxide species has a much lower driving force to conduct HAA, compared to the Cpd I species.120,121 We additionally explored whether ferric-superoxide is capable of initiating HAA from the DKP N7–H bond of the substrate (Figure S23a). However, this process requires a high barrier of over 29.9 kcal/mol (Figure S23b), mainly because the DKP N7–H bond is even stronger than that of the indole N1–H (Figure S23c).27

Figure 10.

Figure 10

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(a) QM(UB3LYP-D3/B1)/MM-optimized ferric-superoxide species in different spin states; the superscripts for OSS, 3, and 5 indicate the open-shell singlet, triplet, and quintet, respectively. The relative energies (RE) are given in kcal/mol and distances in angstroms. (b) QM(UB3LYP-D3/B1)/MM-scanned energy profile for the H-abstraction from OSSRCc. The reaction coordinate is defined as the O10–H1 distance. (c) The calculated BDE values of the N1–H1 bond of (1), the O–H bond of the FeIII–OOH species, and the O–H bond of Cpd II.

4. Conclusions

This study elucidates the molecular mechanism of the P450 NascB-catalyzed dimerization reaction through comprehensive multiscale calculations, as summarized in Scheme 4. The reaction is initiated from HAA from the DKP N7–H atom of Sub1, resulting in the formation of the N7-centered radical species. Subsequently, a pivotal conformational change of the Sub1 radical is necessary to position the N7 atom on the Si-face of the indole C2=C3 double bond. This is followed by intramolecular C2–N7 bond formation to generate a pyrroloindoline radical species. The subsequent intermolecular C3–C6′ bond formation was found to occur via a radical-mediated mechanism, instead of a cationic Friedel–Crafts mechanism. Importantly, the conformational switch of the Sub1 radical not only lowers the barrier of the intermolecular C3–C6′ bond formation but also affords the correct stereoselectivity as observed in experiments. Therefore, our study highlights that the conformational movement of the substrate, which is controlled by the protein environment, can be vital to P450-catalyzed dimerization reactions. We anticipate that these findings can aid our understanding of other P450-catalyzed dimerization reactions, such as intermolecular C–C coupling,122−127 C–N coupling,27,128,129 and C–S coupling.130−133

Scheme 4. QM/MM-Predicted Mechanism of Stereoselective Dimerization by P450 NascB in This Work.

Scheme 4

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Acknowledgments

This work was supported by the National Key Research and Development Program of China (no. 2019YFA0906400 to B.W.) and the National Natural Science Foundation of China (no. 22122305 and 22073077 to B.W. and no. 32025001 to S.L.).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.4c00075.

  • Optimized structures; calculated spin densities; QM/MM energies; and the Cartesian coordinates of all computed species (PDF)

The authors declare no competing financial interest.

Special Issue

Published as part of JACS Auvirtual special issue “Biocatalysis in Asia and Pacific”.

Supplementary Material

au4c00075_si_001.pdf (1.9MB, pdf)

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