Quantum Mechanics/molecular Mechanics Research Articles

In silico modelling of the function of disease-related CAZymes.

In silico modelling of proteins comprises a diversity of computational tools aimed to obtain structural, electronic, and/or dynamic information about these biomolecules, capturing mechanistic details that are challenging to experimental approaches, such as elusive enzyme-substrate complexes, short-lived intermediates, and reaction transition states (TS). The present article gives the reader insight on the use of in silico modelling techniques to understand complex catalytic reaction mechanisms of carbohydrate-active enzymes (CAZymes), along with the underlying theory and concepts that are important in this field. We start by introducing the significance of carbohydrates in nature and the enzymes that process them, CAZymes, highlighting the conformational flexibility of their carbohydrate substrates. Three commonly used in silico methods (classical molecular dynamics (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and enhanced sampling techniques) are described for nonexpert readers. Finally, we provide three examples of the application of these methods to unravel the catalytic mechanisms of three disease-related CAZymes: β-galactocerebrosidase (GALC), responsible for Krabbe disease; α-mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5), involved in cancer; and O-fucosyltransferase 1 (POFUT1), involved in several human diseases such as leukemia and the Dowling-Degos disease.

Investigation of Polymer Aging Mechanisms Using Molecular Simulations: A Review.

Aging has a serious impact on the properties of functional polymers. Therefore, it is necessary to study the aging mechanism to prolong the service and storage life of polymer-based devices and materials. Due to the limitations of traditional experimental methods, more and more studies have adopted molecular simulations to analyze the intrinsic mechanisms of aging. In this paper, recent advances in molecular simulations of the aging of polymers and their composites are reviewed. The characteristics and applications of commonly used simulation methods in the study of the aging mechanisms (traditional molecular dynamics simulation, quantum mechanics, and reactive molecular dynamics simulation) are outlined. The current simulation research progress of physical aging, aging under mechanical stress, thermal aging, hydrothermal aging, thermo-oxidative aging, electric aging, aging under high-energy particle impact, and radiation aging is introduced in detail. Finally, the current research status of the aging simulations of polymers and their composites is summarized, and the future development trend has been prospected.

Effect of the bipodal Ti species on activity and selectivity of propylene epoxidation with H2O2 over TS-1: A theoretical study

In order to probe into the effect of bipodal Ti species on the catalytic performance, the propylene epoxidation with H2O2 over TS-1 catalyst with bipodal Ti site has been studied using an embedded quantum mechanical/molecular mechanics (QM/MM) approach, and the results shown that the bipodal Ti site has strong Brønsted and Lewis acidity, which not only greatly facilitates the epoxidation reaction but also lower selectivity toward the epoxy products. The excellent epoxidation activity is attributed to the Lewis acidity, in which, H2O2 easily activated to generate active intermediates (i.e., Ti-η1-OOH and Ti-η2-OOH) that is facile to transfer O atom to oxidize the propylene. The poor selectivity toward PO is also due to the Lewis acidity, which leads to the stronger adsorption ability of PO and excellent ring-opening ability of Ti-OH nucleophile to PO. The introduction of methoxy (CH3O) group is beneficial for the enhancement of the selectivity of PO, although it enhances the activation free energy for epoxidation to generate PO. The identifications about the relationship of the intrinsic selectivity and activity with bipodal Ti site would provide a clue for designing highly-efficient TS-1 catalysts in propylene epoxidation to PO from the theoretical point of view.

Reaction Mechanism of Glycoside Hydrolase Family 116 Utilizes Perpendicular Protonation.

Retaining glycoside hydrolases use acid/base catalysis with an enzymatic acid/base protonating the glycosidic bond oxygen to facilitate leaving-group departure alongside attack by a catalytic nucleophile to form a covalent intermediate. Generally, this acid/base protonates the oxygen laterally with respect to the sugar ring, which places the catalytic acid/base and nucleophile carboxylates within about 4.5-6.5 Å of each other. However, in glycoside hydrolase (GH) family 116, including disease-related human acid β-glucosidase 2 (GBA2), the distance between the catalytic acid/base and the nucleophile is around 8 Å (PDB: 5BVU) and the catalytic acid/base appears to be above the plane of the pyranose ring, rather than being lateral to that plane, which could have catalytic consequences. However, no structure of an enzyme-substrate complex is available for this GH family. Here, we report the structures of Thermoanaerobacterium xylanolyticum β-glucosidase (TxGH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose and its catalytic mechanism. We confirm that the amide hydrogen bonding to the glycosidic oxygen is in a perpendicular rather than lateral orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations of the glycosylation half-reaction in wild-type TxGH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed 4C1 chair at the -1 subsite. Nevertheless, the reaction can still proceed through a 4H3 half-chair transition state, as in classical retaining β-glucosidases, as the catalytic acid D593 protonates the perpendicular electron pair. The glucose C6OH is locked in a gauche, trans orientation with respect to the C5-O5 and C4-C5 bonds to facilitate perpendicular protonation. These data imply a unique protonation trajectory in Clan-O glycoside hydrolases, which has strong implications for the design of inhibitors specific to either lateral protonators, such as human GBA1, or perpendicular protonators, such as human GBA2.

Chirality of Alanine Molecules in Water Solvent: A Theoretical Perspective

Sequential quantum mechanics/molecular mechanics (QM/MM) calculations combining the average solvent electrostatic configuration (ASEC) and the free energy gradient method are employed to locate minimum structures of α- and β-alanine in a water environment. Herein, we study the solvation effects in the nuclear magnetic resonance (NMR) spectroscopy, vibrational circular dichroism (VCD) spectroscopy, and electronic circular dichroism (ECD) spectroscopy of dl-α-alanine and β-alanine molecules. Our results point out that the ASEC-FEG (average solvent electrostatic configuration with the free energy gradient) method is a suitable approach for finding equilibrium structures of the alanine molecules in aqueous solution. Its accuracy is checked by comparing the optimized structures with those reached by the polarizable continuum model (PCM) and via experimental data. NMR parameters and vibrational and electronic UV-vis spectra are computed with a remarkable agreement with their corresponding experimental values.

Structural Insights into Three Sesquiterpene Synthases for the Biosynthesis of Tricyclic Sesquiterpenes and Chemical Space Expansion by Structure-Based Mutagenesis

The cyclization of farnesyl diphosphate (FPP) into highly strained polycyclic sesquiterpenes is challenging. We here determined the crystal structures of three sesquiterpene synthases (STSs, namely, BcBOT2, DbPROS, and CLM1) catalyzing the biosynthesis of the tricyclic sesquiterpenes presilphiperfolan-8β-ol (1), Δ6-protoilludene (2), and longiborneol (3). All three STS structures contain a substrate mimic, the benzyltriethylammonium cation (BTAC), in their active sites, providing ideal templates for quantum mechanics/molecular mechanics (QM/MM) analyses toward their catalytic mechanisms. The QM/MM-based molecular dynamics (MD) simulations revealed the cascade reactions toward the enzyme products, and different key active site residues that play important roles in stabilizing reactive carbocation intermediates along the three pathways. Site-directed mutagenesis experiments confirmed the roles of these key residues and concomitantly resulted in 17 shunt products (4-20). Isotopic labeling experiments addressed the key hydride and methyl migrations toward the main and several shunt products. These combined methods provided deep insights into the catalytic mechanisms of the three STSs and demonstrated how the chemical space of STSs can rationally be expanded, which may facilitate applications in synthetic biology approaches toward pharmaceutical and perfumery agents.

Water Charge Transfer Accelerates Criegee Intermediate Reaction with H2O- Radical Anion at the Aqueous Interface.

Criegee intermediates (CIs) are important carbonyl oxides that may react with atmospheric trace chemicals and impact the global climate. The CI reaction with water has been widely studied and is a main channel for trapping CIs in the troposphere. Previous experimental and computational reports have largely focused on reaction kinetic processes in various CI-water reactions. The molecular-level origin of CI's interfacial reactivity at the water microdroplet surface (e.g., as found in aerosols and clouds) is unclear. In this study, by employing the quantum mechanical/molecular mechanical (QM/MM) Born-Oppenheimer molecular dynamics with the local second-order Møller-Plesset perturbation theory, our computational results reveal a substantial water charge transfer up to ∼20% per water, which creates the surface H2O+/H2O- radical pairs to enhance the CH2OO and anti-CH3CHOO reactivity with water: the resulting strong CI-H2O- electrostatic attraction at the microdroplet surface facilitates the nucleophilic attack to the CI carbonyl by water, which may counteract the apolar hindrance of the substituent to accelerate the CI-water reaction. Our statistical analysis of the molecular dynamics trajectories further resolves a relatively long-lived bound CI(H2O-) intermediate state at the air/water interface, which has not been observed in gaseous CI reactions. This work provides insights into what may alter the oxidizing power of the troposphere by the next larger CIs than simple CH2OO and implicates a new perspective on the role of interfacial water charge transfer in accelerating molecular reactions at aqueous interfaces.

Computational Modeling of the Interaction of Molecular Oxygen with the miniSOG Protein—A Light Induced Source of Singlet Oxygen

Interaction of molecular oxygen 3O2 with the flavin-dependent protein miniSOG after light illumination results in creation of singlet oxygen 1O2 and superoxide O2●−. Despite the recently resolved crystal structures of miniSOG variants, oxygen-binding sites near the flavin chromophore are poorly characterized. We report the results of computational studies of the protein−oxygen systems using molecular dynamics (MD) simulations with force-field interaction potentials and quantum mechanics/molecular mechanics (QM/MM) potentials for the original miniSOG and the mutated protein. We found several oxygen-binding pockets and pointed out possible tunnels bridging the bulk solvent and the isoalloxazine ring of the chromophore. These findings provide an essential step toward understanding photophysical properties of miniSOG—an important singlet oxygen photosensitizer.

Role of Non-Covalent Interactions in Carbonic Anhydrase I-Topiramate Complex Based on QM/MM Approach.

Carbonic anhydrase (CA) I with a Topiramate (TPM) complex was investigated on the basis of a Quantum Mechanics/Molecular Mechanics (QM/MM) approach. The QM part was treated using Density Functional Theory (DFT) while the MM was simulated using Amberff14SB and GAFF force fields. In addition, the TIP3P model was applied to reproduce the polar environment influence on the studied complex. Next, three snapshots (after 5 ps, 10 ps, and 15 ps of the simulation time) were taken from the obtained trajectory to provide an insight into the non-covalent interactions present between the ligand and binding pocket of the protein. Our special attention was devoted to the binding site rearrangement, which is known in the literature concerning the complex. This part of the computations was performed using ωB97X functional with Grimme D3 dispersion corrections as well as a Becke-Johnson damping function (D3-BJ). Two basis sets were applied: def2-SVP (for larger models) and def2-TZVPD (for smaller models), respectively. In order to detect and describe non-covalent interactions between amino acids of the binding pocket and the ligand, Independent Gradient Model based on Hirshfeld partitioning (IGMH), Interaction Region Indicator (IRI), Quantum Theory of Atoms in Molecules (QTAIM) and Natural Bond Orbitals (NBO) methods were employed. Finally, Symmetry-Adapted Perturbation Theory (SAPT) was applied for energy decomposition between the ligand and protein. It was found that during the simulation time, the ligand position in the binding site was preserved. Nonetheless, amino acids interacting with TPM were exchanging during the simulation, thus showing the binding site reorganization. The energy partitioning revealed that dispersion and electrostatics are decisive factors that are responsible for the complex stability.

Allosteric regulation of β-reaction stage I in tryptophan synthase upon the α-ligand binding.

Tryptophan synthase (TRPS) is a bifunctional enzyme consisting of α- and β-subunits that catalyzes the last two steps of L-tryptophan (L-Trp) biosynthesis. The first stage of the reaction at the β-subunit is called β-reaction stage I, which converts the β-ligand from an internal aldimine [E(Ain)] to an α-aminoacrylate [E(A-A)] intermediate. The activity is known to increase 3-10-fold upon the binding of 3-indole-D-glycerol-3'-phosphate (IGP) at the α-subunit. The effect of α-ligand binding on β-reaction stage I at the distal β-active site is not well understood despite the abundant structural information available for TRPS. Here, we investigate the β-reaction stage I by carrying out minimum-energy pathway searches based on a hybrid quantum mechanics/molecular mechanics (QM/MM) model. The free-energy differences along the pathway are also examined using QM/MM umbrella sampling simulations with QM calculations at the B3LYP-D3/aug-cc-pVDZ level of theory. Our simulations suggest that the sidechain orientation of βD305 near the β-ligand likely plays an essential role in the allosteric regulation: a hydrogen bond is formed between βD305 and the β-ligand in the absence of the α-ligand, prohibiting a smooth rotation of the hydroxyl group in the quinonoid intermediate, whereas the dihedral angle rotates smoothly after the hydrogen bond is switched from βD305-β-ligand to βD305-βR141. This switch could occur upon the IGP-binding at the α-subunit, as evidenced by the existing TRPS crystal structures.

Robust superhydrophobic SiO2/epoxy composite coating prepared by one-step spraying method for corrosion protection of aluminum alloy: Experimental and theoretical studies

In this work, a robust epoxy adhesive (EP) @ superhydrophobic SiO2 (SH-SiO2)-based superhydrophobic coating was prepared on the aluminum substrate by a simple one-step spraying strategy. The prepared EP@SH-SiO2 coating exhibits hierarchical micro/nanostructures and is capable of maintaining a contact angle of more than 150° after 1000 cm of abrasion length and 80 tape-peeling repetitions. Molecular dynamics (MD) simulations and quantum mechanics calculations show that the improved mechanical resistance is attributed to the enhanced interfacial interaction between the EP@SH-SiO2 coating and the underlying substrate induced by the epoxy adhesive. Electrochemical measurements in 3.5 wt% NaCl solution show the corrosion inhibition efficiency of the EP@SH-SiO2 coating reaches up to 99.99%, and even after 8 days of immersion, the low frequency impedance modulus of the EP@SH-SiO2 sample is still ∼1 order of magnitude higher than that bare aluminum, demonstrating excellent long-term corrosion protection. The diffusion behavior of the corrosive medium in the EP@SH-SiO2 coating was revealed by MD simulation to support the experimental results. Additionally, the EP@SH-SiO2 coating exhibits good chemical stability and hydrophobic repair properties. This work will provide a new perspective for the preparation and study of robust superhydrophobic aluminum for corrosion protection properties.

Identification of the protonation and oxidation states of the oxygen-evolving complex in the low-dose X-ray crystal structure of photosystem II.

In photosystem II (PSII), the O3 and O4 sites of the Mn4CaO5 cluster form hydrogen bonds with D1-His337 and a water molecule (W539), respectively. The low-dose X-ray structure shows that these hydrogen bond distances differ between the two homogeneous monomer units (A and B) [Tanaka etal., J. Am Chem. Soc. 2017, 139, 1718]. We investigated the origin of the differences using a quantum mechanical/molecular mechanical (QM/MM) approach. QM/MM calculations show that the short O4-OW539 hydrogen bond (~2.5 Å) of the B monomer is reproduced when O4 is protonated in the S1 state. The short O3-NεHis337 hydrogen bond of the A monomer is due to the formation of a low-barrier hydrogen bond between O3 and doubly-protonated D1-His337 in the overreduced states (S-1 or S-2). It seems plausible that the oxidation state differs between the two monomer units in the crystal.

Mechanism of Chiral-Selective Aminoacylation of an RNA Minihelix Explored by QM/MM Free-Energy Simulations.

Aminoacylation of a primordial RNA minihelix composed of D-ribose shows L-amino acid preference over D-amino acid without any ribozymes or enzymes. This preference in the amino acylation reaction likely plays an important role in the establishment of homochirality in L-amino acid in modern proteins. However, molecular mechanisms of the chiral selective reaction remain unsolved mainly because of difficulty in direct observation of the reaction at the molecular scale by experiments. For seeking a possible mechanism of the chiral selectivity, quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations of the aminoacylation reactions in a modeled RNA were performed to investigate differences in their free-energy profiles along the reactions for L- and D-alanine and its physicochemical origin. The reaction is initiated by approaching a 3'-oxygen of the RNA minihelix to the carbonyl carbon of an aminoacyl phosphate oligonucleotide. The QM/MM umbrella sampling MD calculations showed that the height of the free-energy barrier for L-alanine aminoacylation reaction was 17 kcal/mol, which was 9 kcal/mol lower than that for the D-alanine system. At the transition state, the distance between the negatively charged 3'-oxygen and the positively charged amino group of L-alanine was shorter than that of D-alanine, which was caused by the chirality difference of the amino acid. These results indicate that the transition state for L-alanine is more electrostatically stabilized than that for D-alanine, which would be a plausible mechanism previously unexplained for chiral selectivity in the RNA minihelix aminoacylation.

Multiple Photoisomerization Pathways of the Green Fluorescent Protein Chromophore in a Reversibly Photoswitchable Fluorescent Protein: Insights from Quantum Mechanics/Molecular Mechanics Simulations

Herein, we have employed a combined CASPT2//CASSCF approach within the quantum mechanics/molecular mechanics (QM/MM) framework to explore the early time photoisomerization of rsEGFP2 starting from its two OFF trans states, i.e., Trans1 and Trans2. The results show similar vertical excitation energies to the S1 state in their Franck-Condon regions. Considering the clockwise and counterclockwise rotations of the C11-C9 bond, four pairs of the S1 excited-state minima and low-lying S1/S0 conical intersections were optimized, based on which we determined four S1 photoisomerization paths that are essentially barrierless to the relevant S1/S0 conical intersections leading to efficient excited-state deactivation to the S0 state. Most importantly, our work first identified multiple photoisomerization and excited-state decay paths, which must be seriously considered in the future. This work not only sheds significant light on the primary trans-cis photoisomerization of rsEGFP2 but also aids in the understanding of the microscopic mechanism of GFP-like RSFPs and the design of novel GFP-like fluorescent proteins.

QM/MM Modeling of the Flavin Functionalization in the RutA Monooxygenase

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re-side and si-side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

Water Maintains the UV-Vis Spectral Features During the Insertion of Anionic Naproxen and Ibuprofen into Model Cell Membranes.

UV-vis spectra of anionic ibuprofen and naproxen in a model lipid bilayer of the cell membrane are investigated using computational techniques in combination with a comparative analysis of drug spectra in purely aqueous environments. The simulations aim at elucidating the intricacies behind the negligible changes in the maximum absorption wavelength in the experimental spectra. A set of configurations of the systems constituted by lipid, water, and drugs or just water and drugs are obtained from classical Molecular Dynamics simulations. UV-vis spectra are computed in the framework of atomistic Quantum Mechanical/Molecular Mechanics (QM/MM) approaches together with Time-Dependent Density Functional Theory (TD-DFT). Our results suggest that the molecular orbitals involved in the electronic transitions are the same, regardless of the chemical environment. A thorough analysis of the contacts between the drug and water molecules reveals that no significant changes in UV-vis spectra are a consequence of ibuprofen and naproxen molecules being permanently microsolvated by water molecules, despite the presence of lipid molecules. Water molecules microsolvate the charged carboxylate group as expected but also microsolvate the aromatic regions of the drugs.

Experimental and Computational Study of Aryl-thiosemicarbazones Inhibiting Cruzain Reveals Reversible Inhibition and a Stepwise Mechanism.

Trypanosoma cruzi is a parasite that infects about 6-7 million people worldwide, mostly in Latin America, causing Chagas disease. Cruzain, the main cysteine protease of T. cruzi, is a validated target for developing drug candidates for Chagas disease. Thiosemicarbazones are one of the most relevant warheads used in covalent inhibitors targeting cruzain. Despite its relevance, the mechanism of inhibition of cruzain by thiosemicarbazones is unknown. Here, we combined experiments and simulations to unveil the covalent inhibition mechanism of cruzain by a thiosemicarbazone-based inhibitor (compound 1). Additionally, we studied a semicarbazone (compound 2), which is structurally similar to compound 1 but does not inhibit cruzain. Assays confirmed the reversibility of inhibition by compound 1 and suggested a two-step mechanism of inhibition. The Ki was estimated to be 36.3 μM and Ki* to be 11.5 μM, suggesting the pre-covalent complex to be relevant for inhibition. Molecular dynamics simulations of compounds 1 and 2 with cruzain were used to propose putative binding modes for the ligands. One-dimensional (1D) quantum mechanics/molecular mechanics (QM/MM) potential of mean force (PMF) and gas-phase energies showed that the attack of Cys25-S- on the C═S or C═O bond yields a more stable intermediate than the attack on the C═N bond of the thiosemicarbazone/semicarbazone. Two-dimensional (2D) QM/MM PMF revealed a putative reaction mechanism for compound 1, involving the proton transfer to the ligand, followed by the Cys25-S- attack at C═S. The ΔG and energy barrier were estimated to be -1.4 and 11.7 kcal/mol, respectively. Overall, our results shed light on the inhibition mechanism of cruzain by thiosemicarbazones.

Direct Proton-Coupled Electron Transfer between Interfacial Tyrosines in Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) regulates DNA synthesis and repair in all organisms. The mechanism of Escherichia coli RNR requires radical transfer over a proton-coupled electron transfer (PCET) pathway spanning ∼32 Å across two protein subunits. A key step along this pathway is the interfacial PCET reaction between Y356 in the β subunit and Y731 in the α subunit. Herein, this PCET reaction between two tyrosines across an aqueous interface is explored with classical molecular dynamics and quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The simulations suggest that the water-mediated mechanism involving double proton transfer through an intervening water molecule is thermodynamically and kinetically unfavorable. The direct PCET mechanism between Y356 and Y731 becomes feasible when Y731 is flipped toward the interface and is predicted to be approximately isoergic with a relatively low free energy barrier. This direct mechanism is facilitated by the hydrogen bonding of water to both Y356 and Y731. These simulations provide fundamental insights into radical transfer across aqueous interfaces.

Hybrid Quantum Mechanical/Molecular Mechanical Methods For Studying Energy Transduction in Biomolecular Machines.

Hybrid quantum mechanical/molecular mechanical (QM/MM) methods have become indispensable tools for the study of biomolecules. In this article, we briefly review the basic methodological details of QM/MM approaches and discuss their applications to various energy transduction problems in biomolecular machines, such as long-range proton transports, fast electron transfers, and mechanochemical coupling. We highlight the particular importance for these applications of balancing computational efficiency and accuracy. Using several recent examples, we illustrate the value and limitations of QM/MM methodologies for both ground and excited states, as well as strategies for calibrating them in specific applications. We conclude with brief comments on several areas that can benefit from further efforts to make QM/MM analyses more quantitative and applicable to increasingly complex biological problems.

Dioxygen Binding Is Controlled by the Protein Environment in Non-heme FeII and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme.

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme FeII /2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially bind 2OG by using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off-line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM-MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line FeIII -OO⋅- intermediate in PHF8 and the off-line FeIII -OO⋅- intermediate in EFE.