Figures 2, 3, 4, 5, 6, 7, and 8 show the optimized QM/MM geometries of the QM regions for all relevant minima and transition states (Thr252MeO-Thr mutant, mechanisms I–IV, Glu366 and Asp251 channels). The computed relative QM/MM energies of the stationary points are summarized for basis sets B1/B2 in Table 1 (coupling reaction, mechanisms I and II) and Table 2 (uncoupling reaction, mechanisms III and IV). The single-point energies obtained with the larger TZVP basis (B2) at the corresponding optimized QM/MM geometries (B1) are generally quite similar to those obtained with the smaller basis (B1), although they are consistently slightly higher relative to Compound 0, typically by 1–3 kcal/mol. A similar behavior was also observed for the wild-type enzyme . In the following discussion, we shall only quote B1 results for the sake of consistency (energies, geometries, etc.). Formation of the correct intermediates and products was verified by analysis of the spin densities and Mulliken charges. These data and selected geometrical parameters are documented in the electronic supplementary material.
Mechanism I: homolytic O–O bond cleavage followed by coupled proton–electron transfer
The first step passes over a barrier of 18.1 kcal/mol and leads to an intermediate (IC1), in which the OH moiety forms two hydrogen bonds with MeO-Thr and with Fe=O (Fig. 2). During this step, the Fe–O bond shortens to 1.67 Å in TS1 and then remains at 1.68 Å in IC1. These structural features are similar to those reported for the wild-type enzyme . The spin density and partial charge of the OH group in the first intermediate (IC1) are −0.93 and −0.04, indicating that IC1 contains an OH radical and one-electron-reduced Compound I. This suggests that the O–O bond cleavage is homolytic: the Fe=O moiety carries two unpaired electrons, and the third unpaired electron is mainly located on the OH moiety. IC1 is stabilized by hydrogen-bonding interactions of OH with FeO and MeO-Thr252, and therefore lies only 10.3 kcal/mol above the reactant.
The second step is a hydrogen transfer from the MeO-Thr group to the OH moiety which yields Compound I and water. The corresponding transition state (TS2) lies 18.5 kcal/mol above Compound 0, and the intermediate complex of CH2O-Thr radical with Compound I (IC2) is quite stable, with an energy of 1.1 kcal/mol relative to Compound 0. The OH moiety is obviously reactive enough to abstract a proton from the methoxy group, and the resulting intermediate (IC2) is stabilized by Wat902 and the water molecule formed via two hydrogen bonds.
In the last step, a proton is transported from Glu366 to MeO-Thr in a concerted process via three bridging water molecules. Simultaneously, an electron is transferred from the heme to the methylene group to regenerate the MeO-Thr and form a π cation radical at the heme. The transition state (TS3) and the product (Compound I) lie 17.2 and 8.0 kcal/mol above Compound 0, respectively. The hydrogen-bonding network between Glu366 and MeO-Thr is reoriented after the proton transfer. Overall, the rate-limiting step is the hydrogen abstraction from the methoxy group with a barrier of 18.5 kcal/mol (TS2).
In this channel, the barrier of O–O bond cleavage is 18.6 kcal/mol (TS1 in Fig. 3), similar to the corresponding barrier in the Glu366 channel (18.1 kcal/mol). The intermediate (IC1, OH moiety and one-electron-reduced Compound I) is rather high in energy (14.4 kcal/mol). For the conversion of IC1 to Compound I, a proton needs to be transported from the Asp251 carboxyl group via Wat901 and MeO-Thr to OH, with a concomitant electron transfer from the heme. The spin density and partial charge of the OH group in IC1 are −0.79 and −0.12, indicating that OH will not behave as a “perfect” radical in IC1 owing to the strong hydrogen-bonding interactions with the methoxy group (2.10 Å) and the FeO unit (1.88 Å). In contrast to the wild-type enzyme , the subsequent proton delivery proceeds in two steps. As in the Glu366 channel, a hydrogen atom is first transferred from the methoxy group of MeO-Thr (TS2 at 22.6 kcal/mol, i.e., 8.2 kcal/mol above IC1). The intermediate formed (IC2 at 11.4 kcal/mol) then receives a proton through the Asp251 channel and an electron from the heme in a simultaneous process (TS3 at 23.0 kcal/mol). After releasing its proton, the side chain of Asp251 rotates back into a salt bridge with Arg186, as shown in Fig. 3.
In each channel, the three transition states lie at similar energies relative to Compound 0. The highest point in the reaction profile is TS2 (TS3) in the Glu366 (Asp251) channel at 18.5 (23.0) kcal/mol (see Table 1), i.e., about 4–8 kcal/mol higher than in the wild-type enzyme . The conversion of Compound 0 to Compound I via mechanism I should thus be much slower in the Thr252MeO-Thr mutant compared with the wild-type enzyme.
To ensure that the snapshot used in this study is representative for the system, reaction mechanism I in the Asp251 channel was also studied in an analogous manner with a different snapshot which was drawn after 1,500 ps of MD simulation. The computed relative energies of all stationary points (Table S23) agree with those from the first snapshot (Table 1) to within 1 kcal/mol. The highest point in the reaction profile (TS3) is at 23.2 kcal/mol, very close to the value of 23.0 kcal/mol from the first snapshot (see earlier). The results from both snapshots are thus entirely consistent with each other.
Mechanism II: proton-assisted heterolytic O–O bond cleavage
The energy barrier for direct hydrogen atom transfer from MeO-Thr to FeOOH is 20.8 kcal/mol, and the resulting intermediate (IC1, Fig. 4) lies 0.8 kcal/mol above Compound 0. The unpaired electron is mainly located on the iron atom (iron spin density of 1.37). In contrast to the reaction in the wild-type enzyme, IC1 is not a protonated Compound 0 species, since the O–O bond is cleaved in the first step . However, mechanism II differs from mechanism I, since the hydrogen transfer is part of the first step. In the second step, the concomitant transport of one proton (from Glu366) and one electron (from the heme) leads to formation of Compound I. The relative energies of TS2 and Compound I are 20.0 and 8.0 kcal/mol, respectively.
In the Asp251 channel, we chose several different reaction coordinates to convert Compound 0 to protonated Compound 0 by proton transfer from Asp251 to the distal oxygen atom of the hydroperoxo group. However, all energy scans led to continuously increasing energy profiles, and we were unable to locate protonated Compound 0. Similar problems have also been reported in previous QM/MM calculations for the wild-type enzyme .
Mechanism III: homolytic O1–Fe bond cleavage followed by coupled proton–electron transfer
The optimized geometries are presented in Fig. 5. The barrier (TS1) for homolytic breaking of the O1–Fe bond is 30.3 kcal/mol, and the intermediate (IC1) consisting of iron-bound heme and the OOH radical lies 17.7 kcal/mol above Compound 0. The subsequent hydrogen transfer from MeO-Thr to OOH is very difficult (TS2 at 42.4 kcal/mol, thus 24.7 kcal/mol above IC1), and the second intermediate (IC2) with iron-bound heme and the CH2O-Thr radical is a shallow minimum (IC2 at 40.6 kcal/mol). The barrier for final proton transfer from Glu366 to CH2O-Thr with concomitant electron transfer from the heme is prohibitively high (TS3 at 60.7 kcal/mol). The overall reaction is endothermic by 28.4 kcal/mol.
Figure 6 shows the optimized geometries. In general, the barriers are quite similar to those in the Glu366 channel. The barrier (TS1) for homolytic cleavage of the O1–Fe bond is 28.1 kcal/mol. In the resulting intermediate (IC1 at 25.7 kcal/mol), the spin densities of OOH (−0.97) and iron (1.98) indicate that iron has two unpaired electrons and that OOH is present as a radical. The following hydrogen transfer from MeO-Thr to OOH is again difficult (TS2 at 47.6 kcal/mol, hence 21.9 kcal/mol above IC1) and leads to a very shallow intermediate (IC2 at 47.4 kcal/mol) with a CH2O-Thr radical (spin density of −0.92). The final proton transfer from Asp251 to CH2O-Thr requires much activation (TS3 at 58.4 kcal/mol, i.e., 11.0 kcal/mol above IC2). At the end of the reaction, Asp251 rotates to rebuild the salt bridge with Arg186, as also found in mechanism I. The overall reaction is endothermic by 28.8 kcal/mol.
Mechanism IV: proton-assisted heterolytic O–Fe bond cleavage
As can be seen from Fig. 7, the first step involves O–Fe bond cleavage combined with a hydrogen transfer from MeO-Thr to the proximal oxygen atom. The corresponding barrier is high (TS1 at 40.3 kcal/mol), and the shallow intermediate (IC1 at 38.0 kcal/mol) contains essentially neutral hydrogen peroxide with almost zero spin density and an O1–O2 distance of 1.51 Å; the Fe–O1 distance increases from 1.85 Å (Compound 0) to 3.75 Å (IC1). The subsequent proton transfer from Glu366 to CH2O-Thr again needs much activation (TS2 at 58.3 kcal/mol, thus 20.3 kcal/mol above IC1). The product (ferric resting state and hydrogen peroxide) lies 25.2 kcal/mol above Compound 0.
Figure 8 presents the optimized geometries. The O–Fe bond cleavage with formation of hydrogen peroxide again occurs in the first step, which has a very high barrier (TS1 at 52.9 kcal/mol). The intermediate (IC1 at 47.6 kcal/mol) contains hydrogen peroxide and a CH2O-Thr radical (spin density of −0.90). The transition state for proton transfer from Asp251 to CH2O-Thr (TS2 at 54.9 kcal/mol) lies 7.3 kcal/mol above IC1. The overall reaction is endothermic by 26.4 kcal/mol.