Intermediates in dioxygen activation by methane monooxygenase: a QM/MM study

Abstract

Protein effects in the activation of dioxygen by methane monooxygenase (MMO) were investigated by using combined QM/MM and broken-symmetry Density Functional Theory (DFT) methods. The effects of a novel empirical scheme recently developed by our group on the relative DFT energies of the various intermediates in the catalytic cycle are investigated. Inclusion of the protein leads to much better agreement between the experimental and computed geometric structures for the reduced form (MMOH(red)). Analysis of the electronic structure of MMOH(red) reveals that the two iron atoms have distinct environments. Different coordination geometries tested for the MMOH(peroxo) intermediate reveal that, in the protein environment, the mu-eta2,eta2 structure is more stable than the others. Our analysis also shows that the protein helps to drive reactants toward products along the reaction path. Furthermore, these results demonstrate the importance of including the protein environment in our models and the usefulness of the QM/MM approach for accurate modeling of enzymatic reactions. A discrepancy remains in our calculation of the Fe-Fe distance in our model of HQ as compared to EXAFS data obtained several years ago, for which we currently do not have an explanation.

Figure 1

The catalytic cycle of MMOH. The part of the cycle corresponding to activation of dioxygen is shown in black while the oxidation of the substrate (hydroxylation of methane into methanol in vivo) is in light gray.

Figure 2

Structure of the oxidized MMOH_ox protein (PDB code 1MTY) represented in ribbon. The α subunits are depicted in green, the β subunits in purple and the γ subunits in pink. The QM/MM model used is shown in cartoon representation and is made of a sphere of 35 Å of the α subunit of protomer α (chain D in PDB code 1MTY). The QM part is represented in licorice in the panel (Graphic prepared with VMD82). Solvent-derived ligands are omitted.

Figure 3

Optimized QM/MM structure of Hred (color) superimposed onto the corresponding crystal structure (gray). The first shell atom names are also displayed.

Figure 4

QM/MM structure of Hred including two additional water molecules (color) superimposed onto the standard QM/MM model (gray). The two water molecules have an important effect on the position of Glu 209 and Fe2.

Figure 5

Forces of the protein acting on the QM residues of the active site. On the left, forces correspond to the entire forces (QM forces calculation carried out in vacuum). On the right, forces correspond to what we call “tension forces”, i.e. total forces minus the electrostatic forces of the protein (QM forces calculation with MM charge distribution of the protein included).

Figure 6

Coordination geometries of the two iron atoms in the reduced state. Fe1 is in an octahedral environment, the axial direction of which is defined by the vector Glu 144-O13 (Z₁ vector). Fe2 is in a square pyramidal environment, the axial direction of which is defined by the Fe2-Glu 209 direction (Z₂ vector). Directions X1, Y1 and Z2 are oriented toward the front of the page.

Figure 7

Four tested geometries for H_peroxo. In the upper left, the structure is similar to H_superoxo with a shifted Glu 243 and the peroxide coordinated to iron in a μ-η², η¹ fashion. In the upper right, the peroxide adopts a μ-η²,η² butterfly arrangement. Both structures have been previously studied at the QM level. In the lower part, structures with peroxide coordinated to iron in a cis-μ-1,2 fashion are presented. On the left, the structure is similar to H_superoxo with a shifted Glu 243 (Aμ12H_peroxo), while on the right (Sμ12H_peroxo) it is similar to the μ-η²,η² butterfly peroxo arrangement with Glu 243 having the same conformation as in the crystal structure of the oxidized form.

Figure 8

Geometry of the μ-η²,η² butterfly structure of H_peroxo and different conformers for the Aμ-1,2 H_peroxo structure. The dihedral angle between iron atoms and the peroxide moiety (φ) is also defined (Fe1-O1-O2-Fe2). The first gauche conformation (top left) has a negative angle close to −30 and the second conformer has a positive one close to 40. These two conformers have similar energies and have similar structures as those found in cis-μ-1,2-peroxo model compounds. The last conformer (top right) is closer to the structures found in other cis-μ-1,2-peroxo model compounds.,

Figure 9

Asymmetric structure for H_Q derived from QM/MM calculations.

Figure 10

Schematic reaction energy profile of the QM/MM intermediates as found with B3LYP (gray) and B3LYP-LOC (black).

Figure 11

Destabilization energies (in kcal/mol) due to the protein matrix computed from our QM/MM//RDFT models (Δ[E_QM/MM - E_QM-RODFT] with H_Q taken as a reference in Table S-4 and discussion on the QM/MM//UDFT and QM/MM//RDFT in Supporting Information). The protein matrix effects destabilize more intermediates closer to the reduced form in the reaction pathway.

Scheme 1