Biochemistry than the corresponding B3LYP barriers. The optimized geometries calculated with all three density functionals were almost indistinguishable. As mentioned above, the functional dependence of calculated energy barriers is not uncommon and has been observed previously.58 The exchange correlation functional, which varies between different density functionals, is not systematically improvable. It is therefore important to compare the values of energy barriers calculated with DFT to those obtained with high-level correlated ab initio methods, such as coupled cluster and SCS-MP2.65 The small number of QM atoms required to model the reaction in the active site of sEH allows the use of correlated ab initio quantum chemical methods, such as SCS-MP2, in QM/ MM calculations, which are of a higher level of accuracy and able to include the effects of dispersion in a nonempirical manner. The SCS-MP2 potential energy barriers are higher (by ∼4 kcal/mol) than the corresponding B3LYP barriers. The barriers calculated with the BHandHLYP functional are closest in energy to those calculated with SCS-MP2. This has been found previously in similar calculations on citrate synthase.58 B3LYP has been found to perform reasonably well in the calculation of activation energy barriers for chorismate mutase and p-hydroxybenzoate hydroxylase.65 For chorismate mutase, the authors reported barriers calculated with B3LYP that were lower than those calculated with local coupled cluster theory, LCCSD(T0) (of very high accuracy), by ∼5 kcal/mol. The SCS-MP2//B3LYP/CHARMM22 potential energy barriers (given in the Supporting Information) were used to calculate the corresponding free energy barriers that are listed in Table 3. The SCS-MP2//B3LYP/CHARMM22 free energy barrier for attack at C(1) in t-SO is 5.0 kcal/mol higher than the value calculated with B3LYP/CHARMM22. The SCS- MP2//B3LYP/CHARMM22 barrier of 12.3 kcal/mol for t-SO is in good agreement with the 13−15 kcal/mol range, derived from experiment. The SCS//B3LYP/CHARMM22 barrier calculated for C(1) attack in t-DPPO(1) (12.8 kcal/mol) is also in good agreement with the experimentally derived range of values. In fact, all of the SCS-MP2//B3LYP/CHARMM22 free energy barriers for attack at the preferred carbon atom are within 1 kcal/mol of the estimated experimental barriers. However, it should be emphasized that the quoted range of experimental values is for different substrates in different EH enzymes, and hence, perfect agreement should not be expected. A summary of the agreement between experiment and theory for the t-DPPO(1) model is shown in Table 4. It should be emphasized also that the excellent agreement between calculations and experiment is only observed in this work where the highest level of QM theory (SCS-MP2) is used. Despite this, B3LYP (and AM1) do predict the correct regioselectivity and are hence suitable for qualitative predictions of reactivity. This has also been found to be true for other enzymes.65 While B3LYP underestimates barrier heights in this example, in calculations for citrate synthase, it was found to overestimate barriers.58 Hydrogen Bonding Analysis. As mentioned above, there are several important hydrogen bonding interactions present in the active site of sEH. Hydrogen bonding analysis was performed on all umbrella sampling MD simulations (using CHARMM), to gain insight into the lifetimes of these interactions and how they vary during the nucleophilic attack and to assess their effect on the calculated reaction barriers. A hydrogen bond was defined as being present when the distance between the donor (D) and acceptor (A) heavy atoms was less than 2.8 Å and the A−H−D angle was greater than 90°. This definition has been used in Article previous work.82 Detailed data are given in the Supporting Information, and the most interesting features are described below. In all of the MD simulations in which the His523 residue is protonated at the Nε position, the Nε proton is involved in a hydrogen bond with the Asp333 Oδ2 atom, which is conserved throughout the simulations. The Asp333 Oδ1 atom (the carboxylate oxygen atom that does not act as the nucleophile) accepts hydrogen bonds from the backbone NH groups of Phe265 and Trp334. These hydrogen bonds have been identified as being important in stabilizing the negative charge that accumulates on the Asp333 Oδ1 atom upon formation of the ester intermediate.33,50 A hydrogen bond is formed between the epoxide oxygen atom and the hydroxyl group of Tyr381 for the entire duration of all umbrella sampling MD simulations, in the ring-opened and closed forms of the substrate. This hydrogen bond becomes stronger as the reaction progresses, as signified by a decrease in the distance between the epoxide oxygen atom and hydroxyl proton of Tyr381 of ∼0.2 Å. The hydrogen bond between the epoxide oxygen and the Tyr465 hydroxyl group shows more variance, as is discussed below. Tyr381 and Tyr465 have both been identified as being involved in the catalytic mechanism of sEH, by stabilization of the oxyanion hole formed after nucleophilic attack.33−36 It has not been conclusively established whether these residues are directly involved in any bond-breaking or -forming processes. As Tyr381 and Tyr465 were not included in the QM region in this work, it was not possible to observe protonation of the ring-opened epoxide by either of these residues. However, the excellent agreement between our high-level (SCS-MP2) free energy barriers and those derived from experimental rate constants supports a mechanism in which neither of the active site tyrosine residues is deprotonated. Instead, it is believed that the tyrosine residues act as hydrogen bond donors, as found with glutathione S-transferase M1-1.82 In the t-SO umbrella sampling MD simulations of nucleophilic attack at C(2), a hydrogen bond is not observed between the Tyr465 hydroxyl group and the epoxide oxygen in the TS for the majority of the simulations (Figure 5). This Figure 5. Active site structures taken from AM1/CHARMM22 umbrella sampling MD simulations, corresponding to the transition states for the reaction of t-SO. The blue and orange structures correspond to nucleophilic attack at C(1) and C(2), respectively. 1781 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786