Substrate hydroxylation in methane monooxygenase: quantitative modeling via mixed quantum mechanics/molecular mechanics techniques

Using broken-symmetry unrestricted density functional theory quantum mechanical (QM) methods in concert with mixed quantum mechanics/molecular mechanics (QM/MM) methods, the hydroxylation of methane and substituted methanes by intermediate Q in methane monooxygenase hydroxylase (MMOH) has been quantitatively modeled. This protocol allows the protein environment to be included throughout the calculations and its effects (electrostatic, van der Waals, strain) upon the reaction to be accurately evaluated. With the current results, recent kinetic data for CH3X (X = H, CH3, OH, CN, NO2) substrate hydroxylation in MMOH (Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 8770-8771) can be rationalized. Results for methane, which provide a quantitative test of the protocol, including a substantial kinetic isotope effect (KIE), are in reasonable agreement with experiment. Specific features of the interaction of each of the substrates with MMO are illuminated by the QM/MM modeling, and the resulting effects upon substrate binding are quantitatively incorporated into the calculations. The results as a whole point to the success of the QM/MM methodology and enhance our understanding of MMOH catalytic chemistry. We also identify systematic errors in the evaluation of the free energy of binding of the Michaelis complexes of the substrates, which most likely arise from inadequate sampling and/or the use of harmonic approximations to evaluate the entropy of the complex. More sophisticated sampling methods will be required to achieve greater accuracy in this aspect of the calculation.     

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

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.     

Structure, electronic configuration, and Mössbauer spectral parameters of an antiferromagnetic Fe2-peroxo intermediate of methane monooxygenase

Determining structures of reaction intermediates is crucial for understanding catalytic cycles of metalloenzymes. However, short life times or experimental difficulties have prevented obtaining such structures for many enzymes of interest. We report geometric and electronic structures of a peroxo intermediate in the catalytic cycle of methane monooxygenase hydroxylase (MMOH) for which there is no crystallographic characterization. The structure was predicted via spin density functional theory using (57)Fe M?ssbauer spectral parameters as a reference. Computed isomer shifts (δ(Fe) = +0.68, +0.66 mm s(-1)) and quadrupole splittings (ΔE(Q) = -1.49, -1.48 mm s(-1)) for the predicted structure are in excellent agreement with experimental values of a peroxo MMOH intermediate. Predicted peroxo to iron charge transfer bands agree with UV-Vis spectroscopy. Peroxide binds in a cis μ-1,2 fashion and plays a dominant role in the active site's electronic structure. This induces a ferromagnetic to antiferromagnetic transition of the diiron core weakening the O-O bond in preparation for cleavage in subsequent steps of the catalytic cycle.     

X-ray crystal structures of manganese(II)-reconstituted and native toluene/o-xylene monooxygenase hydroxylase reveal rotamer shifts in conserved residues and an enhanced view of the protein interior

We report the X-ray crystal structures of native and manganese(II)-reconstituted toluene/o-xylene monooxygenase hydroxylase (ToMOH) from Pseudomonas stutzeri OX1 to 1.85 and 2.20 A resolution, respectively. The structures reveal that reduction of the dimetallic active site is accompanied by a carboxylate shift and alteration of the coordination environment for dioxygen binding and activation. A rotamer shift in a strategically placed asparagine 202 accompanies dimetallic center reduction and is proposed to influence protein component interactions. This rotamer shift is conserved between ToMOH and the corresponding residue in methane monooxygenase hydroxylase (MMOH). Previously unidentified hydrophobic pockets similar to those present in MMOH are assigned.     

Effects of the protein environment on the structure and energetics of active sites of metalloenzymes. ONIOM study of methane monooxygenase and ribonucleotide reductase.

As the first application of our recently developed ONIOM2(QM:MM) and ONIOM3(QM:QM:MM) codes to the metalloenzymes with a large number of protein residues, two members of the non-heme protein family, methane monooxygenause and ribonucleotide reductase, have been chosen. The "active-site + four alpha-helical fragments" model was adopted which includes about 1000 atoms from 62 residues around the Fe-centered spheres. Comparison of the active-site geometries of MMOH and R2 units optimized with this model with those obtained with the "active site only" (with only 39-46 atoms) model and the X-ray results clearly demonstrates the crucial role of the active site-protein interaction in the enzymatic activities.     

Reactions of the peroxo intermediate of soluble methane monooxygenase hydroxylase with ethers

Soluble methane monooxygenase (sMMO) isolated from Methylococcus capsulatus (Bath) utilizes a carboxylate-bridged diiron center and dioxygen to catalyze the conversion of methane to methanol. Previous studies revealed that a di(mu-oxo)diiron(IV) intermediate termed Q is responsible for the catalytic activity with hydrocarbons. In addition, the peroxodiiron(III) intermediate (H(peroxo)) that precedes Q formation in the catalytic cycle has been demonstrated to react with propylene, but its reactivity has not been extensively investigated. Given the burgeoning interest in the existence of multiple oxidants in metalloenzymes, a more exhaustive study of the reactivity of H(peroxo) was undertaken. The kinetics of single turnover reactions of the two intermediates with ethyl vinyl ether and diethyl ether were monitored by single- and double-mixing stopped-flow optical spectroscopy. For both substrates, the rate constants for reaction with H(peroxo) are greater than those for Q. An analytical model for explaining the transient kinetics is described and used successfully to fit the observed data. Activation parameters were determined through temperature-dependent studies, and the kinetic isotope effects for the reactions with diethyl ether were measured. The rate constants indicate that H(peroxo) is a more electrophilic oxidant than Q. We propose that H(peroxo) reacts via two-electron transfer mechanisms, and that Q reacts by single-electron transfer steps.     

X-ray absorption spectroscopic study of the reduced hydroxylases of methane monooxygenase and toluene/o-xylene monooxygenase: differences in active site structure and effects of the coupling proteins MMOB and ToMOD

The diiron active sites of the reduced hydroxylases from methane monooxygenase (MMOH(red)) and toluene/o-xylene monooxygenase (ToMOH(red)) have been investigated by X-ray absorption spectroscopy (XAS). Results of Fe K-edge and extended X-ray absorption fine structure analysis reveal subtle differences between the hydroxylases that may be correlated to access of the active site. XAS data were also recorded for each hydroxylase in the presence of its respective coupling protein. MMOB affects the outer-shell scattering contributions in the diiron site of MMOH(red), whereas ToMOD exerts its main effect on the first-shell ligation of ToMOH(red); it also causes a slight decrease in the Fe-Fe separation. These results provide an initial step toward delineating the differences in structure and reactivity in bacterial multicomponent monooxygenase proteins.     

Modeling of phytochrome absorption spectra

Phytochromes constitute one of the six well‐characterized families of photosensory proteins in Nature. From the viewpoint of computational modeling, however, phytochromes have been the subject of much fewer studies than most other families of photosensory proteins, which is likely a consequence of relevant high‐resolution structural data becoming available only in recent years. In this work, hybrid quantum mechanics/molecular mechanics (QM/MM) methods are used to calculate UV‐vis absorption spectra of Deinococcus radiodurans bacteriophytochrome. We investigate how the choice of QM/MM methodology affects the resulting spectra and demonstrate that QM/MM methods can reproduce the experimental absorption maxima of both the Q and Soret bands with an accuracy of about 0.15 eV. Furthermore, we assess how the protein environment influences the intrinsic absorption of the bilin chromophore, with particular focus on the Q band underlying the primary photochemistry of phytochromes. © 2013 Wiley Periodicals, Inc.     

Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit

The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB.     

QM/MM study of the taxadiene synthase mechanism

Combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the reaction mechanism of taxadiene synthase (TXS). TXS catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to taxadiene (T) and four minor cyclic products. All these products originate from the deprotonation of carbocation intermediates. The reaction profiles for the conversion of GGPP to T as well as to minor products were calculated for different configurations of relevant TXS carbocation complexes. The QM region was treated at the M06-2X/TZVP level, while the CHARMM27 force field was used to describe the MM region. The QM/MM calculations suggest a reaction pathway for the conversion of GGPP to T, which slightly differs from previous proposals regarding the number of reaction steps and the conformation of the carbocations. The QM/MM results also indicate that the formation of minor products via water-assisted deprotonation of the carbocations is highly exothermic, by about −7 to −23 kcal/mol. Curiously, however, the computed barriers and reaction energies indicate that the formation of some of the minor products is more facile than the formation of T. Thus, the present QM/MM calculations provide detailed insights into possible reaction pathways and into the origin of the promiscuity of TXS, but they do not reproduce the product distribution observed experimentally. © 2019 Wiley Periodicals, Inc.     

Characterization of the arene-oxidizing intermediate in ToMOH as a diiron(III) species

We report the generation and characterization of a diiron(III) intermediate formed during reaction with dioxygen of the reduced hydroxylase component of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1. The decay rate of this species is accelerated upon mixing with phenol, a substrate for this system. Under steady-state conditions, hydrogen peroxide was generated in the absence of substrate. The oxidized hydroxylase also decomposed hydrogen peroxide to liberate dioxygen in the absence of reducing equivalents. This activity suggests that dioxygen activation may be reversible. The linear free energy relationship determined from hydroxylation of para-substituted phenols under steady-state turnover has a negative slope. A value of rho < 0 is consistent with electrophilic attack by the oxidizing intermediate on the aromatic substrates. The results from these steady and pre-steady-state experiments provide compelling evidence that the diiron(III) intermediate is the active oxidant in the toluene/o-xylene monooxygenase system and is a peroxodiiron(III) transient, despite differences between its optical and M?ssbauer spectroscopic parameters and those of other peroxodiiron(III) centers.     

Ligand rotation in [Ar(R)N]3M-N2-M'[N(R)Ar]3(M, M' = MoIII, NbIII; R = iPr and tBu) dimers

Earlier calculations on the model N2-bridged dimer (micro-N2)-{Mo[NH2]3}2 revealed that ligand rotation away from a trigonal arrangement around the metal centres was energetically favourable resulting in a reversal of the singlet and triplet energies such that the singlet state was stabilized 13 kJ mol(-1) below the D(3d) triplet structure. These calculations, however, ignored the steric bulk of the amide ligands N(R)Ar (R =iPr and tBu, Ar = 3,5-C6H3Me2) which may prevent or limit the extent of ligand rotation. In order to investigate the consequences of steric crowding, density functional calculations using QM/MM techniques have been performed on the Mo(III)Mo(III) and Mo(III)Nb(III) intermediate dimer complexes (mu-N(2))-{Mo[N(R)Ar]3}2 and [Ar(R)N]3Mo-(mu-N2)-Nb[N(R)Ar]3 formed when three-coordinate Mo[N(R)Ar]3 and Nb[N(R)Ar]3 react with dinitrogen. The calculations indicate that ligand rotation away from a trigonal arrangement is energetically favourable for all of the ligands investigated and that the distortion is largely electronic in origin. However, the steric constraints of the bulky amide groups do play a role in determining the final orientation of the ligands, in particular, whether the ligands are rotated at one or both metal centres of the dimer. Analogous to the model system, QM/MM calculations predict a singlet ground state for the (mu-N2)-{Mo[N(R)Ar]3}2 dimers, a result which is seemingly at odds with the experimental triplet ground state found for the related (mu-N2)-{Mo[N(tBu)Ph]3}2 system. However, QM/MM calculations on the (mu-N2)-{Mo[N(tBu)Ph]3}2 dimer reveal that the singlet-triplet gap is nearly 20 kJ mol(-1) smaller and therefore this complex is expected to exhibit very different magnetic behaviour to the (mu-N2)-{Mo[N(R)Ar]3}2 system.     

Fragmentation-based QM/MM simulations: length dependence of chain dynamics and hydrogen bonding of polyethylene oxide and polyethylene in aqueous solutions

Molecular fragmentation quantum mechanics (QM) calculations have been combined with molecular mechanics (MM) to construct the fragmentation QM/MM method for simulations of dilute solutions of macromolecules. We adopt the electrostatics embedding QM/MM model, where the low-cost generalized energy-based fragmentation calculations are employed for the QM part. Conformation energy calculations, geometry optimizations, and Born-Oppenheimer molecular dynamics simulations of poly(ethylene oxide), PEO(n) (n = 6-20), and polyethylene, PE(n) ( n = 9-30), in aqueous solution have been performed within the framework of both fragmentation and conventional QM/MM methods. The intermolecular hydrogen bonding and chain configurations obtained from the fragmentation QM/MM simulations are consistent with the conventional QM/MM method. The length dependence of chain conformations and dynamics of PEO and PE oligomers in aqueous solutions is also investigated through the fragmentation QM/MM molecular dynamics simulations.     

Dioxygen activation in methane monooxygenase: a theoretical study

Using broken-symmetry unrestricted Density Functional Theory, the mechanism of enzymatic dioxygen activation by the hydroxylase component of soluble methane monooxygenase (MMOH) is determined to atomic detail. After a thorough examination of mechanistic alternatives, an optimal pathway was identified. The diiron(II) state H(red) reacts with dioxygen to give a ferromagnetically coupled diiron(II,III) H(superoxo) structure, which undergoes intersystem crossing to the antiferromagnetic surface and affords H(peroxo), a symmetric diiron(III) unit with a nonplanar mu-eta(2):eta(2)-O(2)(2)(-) binding mode. Homolytic cleavage of the O-O bond yields the catalytically competent intermediate Q, which has a di (mu-oxo)diiron(IV) core. A carboxylate shift involving Glu243 is essential to the formation of the symmetric H(peroxo) and Q structures. Both thermodynamic and kinetic features agree well with experimental data, and computed spin-exchange coupling constants are in accord with spectroscopic values. Evidence is presented for pH-independent decay of H(red) and H(peroxo). Key electron-transfer steps that occur in the course of generating Q from H(red) are also detailed and interpreted. In contrast to prior theoretical studies, a requisite large model has been employed, electron spins and couplings have been treated in a quantitative manner, potential energy surfaces have been extensively explored, and quantitative total energies have been determined along the reaction pathway.     

Structural model studies for the peroxo intermediate P and the reaction pathway from P-->Q of methane monooxygenase using broken-symmetry density functional calculations

Several structural models for the active site of the peroxo intermediate state "P" of the hydroxylase component of soluble methane monooxygenase (MMOH) have been studied, using two DFT functionals OPBE and PW91 with broken-symmetry methodology and the conductor-like screening (COSMO) solvation model. These active site models have different O2 binding modes to the diiron center, such as the mu-eta2,eta2, trans-mu-1,2 and cis-mu-1,2 conformations. The calculated properties, including optimized geometries, electronic energies, Fe net spin populations, and M?ssbauer isomer shift and quadrupole splitting values, have been reported and compared with available experimental results. The high-spin antiferromagnetically (AF) coupled Fe3+ sites are correctly predicted by both OPBE and PW91 methods for all active site models. Our data analysis and comparisons favor a cis-mu-1,2 structure (model cis-mu-1,2a shown in Figure 9) likely to represent the active site of MMOH-P. Feasible structural changes from MMOH-P to another intermediate state MMOH-Q are also proposed, where the carboxylate group of Glu243 side chain has to open up from the mono-oxygen bridging position, and the dissociations of the terminal H2O ligand from Fe1 and of the oxygen atom in the carboxylate group of Glu144 from Fe2 are also necessary for the O2 binding mode changes from cis to trans. The O-O bond is proposed to break in the trans-conformation and forms two mu-oxo bridges in MMOH-Q. The terminal H2O molecule and the Glu144 side chain then rebind with Fe1 and Fe2, respectively, in Q.     

Protein/ligand binding free energies calculated with quantum mechanics/molecular mechanics

The calculation of binding affinities for flexible ligands has hitherto required the availability of reliable molecular mechanics parameters for the ligands, a restriction that can in principle be lifted by using a mixed quantum mechanics/molecular mechanics (QM/MM) representation in which the ligand is treated quantum mechanically. The feasibility of this approach is evaluated here, combining QM/MM with the Poisson-Boltzmann/surface area model of continuum solvation and testing the method on a set of 47 benzamidine derivatives binding to trypsin. The experimental range of the absolute binding energy (DeltaG = -3.9 to -7.6 kcal/mol) is reproduced well, with a root-mean-square (RMS) error of 1.2 kcal/mol. When QM/MM is applied without reoptimization to the very different ligands of FK506 binding protein the RMS error is only 0.7 kcal/mol. The results show that QM/MM is a promising new avenue for automated docking and scoring of flexible ligands. Suggestions are made for further improvements in accuracy.     

Reversible O-O Bond Scission of Peroxodiiron(III) to High-Spin Oxodiiron(IV) in Dioxygen Activation of a Diiron Center with a Bis-tpa Dinucleating Ligand as a Soluble Methane Monooxygenase Model

The conversion of peroxodiiron(III) to high-spin S = 2 oxodiiron(IV) via reversible O-O bond scission in a diiron complex with a bis-tpa dinucleating ligand, 6-hpa, has been characterized by elemental analysis; kinetic measurements for alkene epoxidation; cold-spray ionization mass spectrometry; and electronic absorption, M?ssbauer, and resonance Raman spectroscopy to gain insight into the O(2) activation mechanism of soluble methane monooxygenases. This is the first synthetic example of a high-spin S = 2 oxodiiron(IV) species that oxidizes alkenes to epoxides efficiently. The bistability of the peroxodiiron(III) and high-spin S = 2 oxodiiron(IV) moieties is the key feature for the reversible O-O bond scission.     

The prediction of Fe Mössbauer parameters by the density functional theory: a benchmark study

We report the performance of eight density functionals (B3LYP, BPW91, OLYP, O3LYP, M06, M06-2X, PBE, and SVWN5) in two Gaussian basis sets (Wachters and Partridge-1 on iron atoms; cc-pVDZ on the rest of atoms) for the prediction of the isomer shift (IS) and the quadrupole splitting (QS) parameters of M?ssbauer spectroscopy. Two sources of geometry (density functional theory-optimized and X-ray) are used. Our data set consists of 31 iron-containing compounds (35 signals), the M?ssbauer spectra of which were determined at liquid helium temperature and where the X-ray geometries are known. Our results indicate that the larger and uncontracted Partridge-1 basis set produces slightly more accurate linear correlations of electronic density used for the prediction of IS and noticeably more accurate results for the QS parameter. We confirm and discuss the earlier observation of Noodleman and co-workers that different oxidation states of iron produce different IS calibration lines. The B3LYP and O3LYP functionals have the lowest errors for either IS or QS. BPW91, OLYP, PBE, and M06 have a mixed success whereas SVWN5 and M06-2X demonstrate the worst performance. Finally, our calibrations and conclusions regarding the best functional to compute the M?ssbauer characteristics are applied to candidate structures for the peroxo and Q intermediates of the enzyme methane monooxygenase hydroxylase (MMOH), and compared to experimental data in the literature.     

Computational methods for the study of enzymic reaction mechanisms III: a perturbation plus QM/MM approach for calculating relative free energies of protonation

We describe a coupling parameter, that is, perturbation, approach to effectively create and annihilate atoms in the quantum mechanical Hamiltonian within the closed shell restricted Hartree-Fock formalism. This perturbed quantum mechanical atom (PQA) method is combined with molecular mechanics (MM) methods (PQA/MM) within a molecular dynamics simulation, to model the protein environment (MM region) effects that also make a contribution to the overall free energy change. Using the semiempirical PM3 method to model the QM region, the application of this PQA/MM method is illustrated by calculation of the relative protonation free energy of the conserved OD2 (Asp27) and the N5 (dihydrofolate) proton acceptor sites in the active site of Escherichia coli dihydrofolate reductase (DHFR) with the bound nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. For a number of choices for the QM region, the relative protonation free energy was calculated as the sum of contributions from the QM region and the interaction between the QM and MM regions via the thermodynamic integration (TI) method. The results demonstrate the importance of including the whole substrate molecule in the QM region, and the overall protein (MM) environment in determining the relative stabilities of protonation sites in the enzyme active site. The PQA/MM free energies obtained by TI were also compared with those estimated by a less computationally demanding nonperturbative method based on the linear response approximation (LRA). For some choices of QM region, the total free energies calculated using the LRA method were in very close agreement with the PQA/MM values. However, the QM and QM/MM component free energies were found to differ significantly between the two methods.