Biochemistry hydrogen bond is present in the simulations of C(1) attack that gave the lowest barriers, but it is not present in those with higher energy barriers (details in the Supporting Information). When not donating a hydrogen bond to the epoxide oxygen atom, the hydroxyl group of Tyr465 in the protonated His523 model points toward the phenyl group of Phe265 and is not involved in any hydrogen bonding. The Tyr465−epoxide oxygen hydrogen bond does not form in any of the simulations with the neutral His523 model, because the hydroxyl group of Tyr465 instead donates a hydrogen bond to the Asp333 Oδ2 atom, as shown in Figure 3. The ability of Tyr465 to form a hydrogen bond to the Asp333 Oδ2 atom (when His523 is neutral) may be significant for the proton shuttle step during hydrolysis. In the simulations of nucleophilic attack at C(2) of t-SO, the hydrogen bond between the backbone NH group of Phe265 and Oδ1 of Asp333 is only present for 50% of the simulation time, after r = −0.2 Å (corresponding to the TS). In all of the t-DPPO(1) simulations in which His523 is protonated, a hydrogen bond is present between the hydroxyl group of Tyr465 and the epoxide oxygen for most of the time, throughout the reaction, for nucleophilic attack at C(1) and C(2). This explains the lower barriers that are observed for the t-DPPO(1) simulations, compared to those of t-SO (Table 3), Article where this interaction is only present for a fraction of the simulation time. In gas phase single-point B3LYP/6-31G(d) energy calculations (details below), the “intrinsic” barriers to C(1) attack in t-SO and t-DPPO were found to be similar. The hydrogen bond between Tyr465 and the epoxide oxygen is not always present in the t-DPPO(2) simulations, for attack at C(1) or C(2), as shown in Figure 6. The transition state (free energy maximum) is located at r = −0.2 Å in both cases. The hydrogen bond analysis, together with the free energy barriers in Table 3, indicates that the hydrogen bond between the hydroxyl group of Tyr465 and the epoxide oxygen atom plays a significant role in lowering the barrier to nucleophilic attack in t-SO: higher barriers for nucleophilic attack at C(1) were observed when this interaction was not present in the TS (see the Supporting Information for details). This interaction was not observed during attack at C(2) for t-SO, which is likely to explain the difference between the barrier for attack at C(1) and C(2). The binding mode of t-SO in murine sEH may be an additional factor contributing to the lower barrier for attack at C(1). The O−C(1) distance is 0.86 Å shorter than the O− C(2) distance, on average, at the end of unrestrained simulations of t-SO (Table 1). Considering that the two Figure 6. Hydrogen bonding to the epoxide oxygen atom during nucleophilic attack at (a) C(1) of t-SO, (b) C(2) of t-SO, (c) C(1) of t-DPPO(1), (d) C(2) of t-DPPO(1), (e) C(1) of t-DPPO(2), and (f) C(2) of t-DPPO(2). The height of the graph corresponds to the total number of hydrogen bonds to the epoxide oxygen, n(HB), at each value of r, during the extended AM1/CHARMM22 umbrella sampling MD simulations, averaged over all profiles. The contributions from Tyr381, Tyr465, and Trp334 are colored blue, red, and green, respectively. The reactant complex is at r = −2.0 Å; the alkyl−enzyme intermediate is at r = 0.5 Å, and the transition state is around r = −0.2 Å (see the text for the definition of r). 1782 dx.doi.org/10.1021/bi201722j | Biochemistry 2012, 51, 1774−1786