Biochemistry single-point energy calculations at the B3LYP/6-311+G(d,p) level on the B3LYP/6-31G(d)/CHARMM22 geometries. Ab initio QM/MM potential energy profiles were calculated using MOLPRO,71 by performing single-point energy calculations on the QM region taken from the B3LYP/CHARMM22 geo- metries and including the point charges from the MM region in the QM Hamiltonian. B3LYP/CHARMM22 geometries have been used previously for higher-level single-point energies and were found to perform well at predicting geometries in other systems. Ab initio QM energies were obtained using spin- component-scaled second-order Møller−Plesset perturbation theory (SCS-MP2),72 with the correlation-consistent polarized valence triple-ζ basis set (cc-pVTZ) developed by Dunning and co-workers.73−75 Additional diffuse functions were included on the epoxide oxygen atom (aug-cc-pVTZ). When a small basis set is employed, SCS-MP2 calculations can be less accurate than B3LYP calculations with the same basis set. However, SCS-MP2 is usually more accurate than B3LYP when a larger basis set is used, such as the one used in this work.58,76 SCS-MP2 is a correlated ab initio method and hence the most accurate QM method of those used here and has been shown to calculate values of energy barriers within 0.5 kcal/mol of those calculated with the LCCSD(T) coupled cluster method (with the same basis set).58 LCCSD(T) QM/MM calculations have been shown to give energy barriers that approach chemical accuracy (1 kcal/mol). Only one SCS-MP2//B3LYP/CHARMM22 barrier was computed for C(1) and C(2) attack in each substrate model, because of the computational cost. ■ RESULTS AND DISCUSSION Unrestrained QM/MM MD Simulations. The active site structures from the 15 ps unrestrained QM/MM MD simulations performed prior to umbrella sampling simulations were very similar (in terms of interatomic distances, hydrogen bonding, etc.) for each enzyme−substrate model system. A representative structure from the t-DPPO(1) simulation is shown in Figure 3. The Nε atom of His523 is protonated in Figure 3. Representative structure from an AM1/CHARMM22 umbrella sampling MD simulation (at r = −2.0 Å) of the t-DPPO(1) system, showing important residues. The His523 residue is protonated at the Nε atom in the model shown here and forms a hydrogen bond with the Oδ2 atom of Asp333. this structure (also see below). There are several important interactions common to all three enzyme−substrate model systems. These include hydrogen bonds between the epoxide oxygen atom of each substrate and the hydroxyl groups of Tyr381 and Tyr465 and a hydrogen bond between the Nε Article hydrogen of His523 and the Oδ2 atom of Asp333. The latter interaction is absent in the enzyme−substrate models in which the His523 Nε atom is not protonated. The values of the interatomic distances involved in the definition of the reaction coordinate, averaged over the 10 starting structures for each model system, are listed in Table 1. The substrate orientation in Table 1. Average Oδ2−C(x) Distances (in angstroms) Calculated from the Structures from the End of Each of Ten 15 ps Unrestrained QM/MM MD Simulations Performed for the t-SO, t-DPPO(1), and t-DPPO(2) Systems model system t-SO t-DPPO(1) t-DPPO(2) Oδ2−C(1) 2.91 ± 0.27 3.21 ± 0.38 2.76 ± 0.17 Oδ2−C(2) 3.77 ± 0.44 3.32 ± 0.24 3.40 ± 0.25 the active site appears to favor nucleophilic attack by the Oδ2 atom of Asp333 at C(1) for both the t-SO and t-DPPO(2) models, as the Oδ2−C(1) distances are significantly shorter than the Oδ2−C(2) distances (by >0.6 Å). The C(1) atom in t-DPPO corresponds to the phenyl epoxide carbon (as shown in Figure 1). The distinction is less clear for the t-DPPO(1) model; the orientation of the substrate does not suggest a preference in the regioselectivity of nucleophilic attack, as the difference between the Oδ2−C(1) and Oδ2−C(2) distances is small (∼0.1 Å). Protonation State of His523. As mentioned in the introductory section, the protonation state of His523 responsible for activity remains a subject of debate.37,50 Free energy profiles were obtained from preliminary AM1/ CHARMM22 umbrella sampling MD simulations, in which 10 ps of MD was performed at each value of the reaction coordinate (r). The free energy barriers for the reaction, calculated by combining the trajectories from the five simulations for each system, are listed in Table 2. The barriers Table 2. Activation Free Energies, ΔG⧧ (in kilocalories per mole) for the Nucleophilic Ring-Opening Reaction at the C(1) and C(2) Atoms of the Epoxide Substrate in the Enzyme−Substrate Models t-SO, t-DPPO(1), and t-DPPO(2)a ΔG⧧(P) ΔG⧧(N) model system C(1) C(2) C(1) C(2) t-SO 21.8 33.2 32.1 31.8 t-DPPO(1) 23.5 24.5 29.3 26.5 t-DPPO(2) 18.4 27.6 28.9 27.0 aAll energies were calculated by combining the trajectories of five preliminary AM1/CHARMM22 umbrella sampling MD simulations, with 10 ps of dynamics simulated at each value of r. P denotes the enzyme model in which the His523 Nε atom is protonated (and His523 is positively charged). N denotes the enzyme model in which the His523 Nε atom is not protonated (and His523 is neutral). to nucleophilic attack at C(1) in all three sEH models studied [t-SO, t-DPPO(1), and t-DPPO(2)] are significantly higher when His523 is neutral (N) than when it is protonated (P). The barriers for attack at C(2) are largely unaffected by the protonation state of His523. Consequently, the regioselectivity of the ring-opening reaction is significantly affected by the protonation state of His523; the relative barriers suggest a 1778 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786