Combined computational and crystallographic study of the oxidised states of [NiFe] hydrogenase

[NiFe] hydrogenases catalyse the reaction H₂↔2H⁺+2e⁻. Several states of the enzyme have been observed by spectroscopic methods. Among these, the two most oxidized states, called the unready Ni–A and Ni–SU states, have been especially intriguing, because they take a much longer time to activate than the corresponding ready Ni–B and Ni–SI states. It has recently been suggested that the unready states actually contain a (hydro)peroxide bridge between the Ni and Fe ions, in contrast to the hydroxide bridge in the ready states. In this paper, we use quantum refinement (crystallographic refinement, in which the molecular mechanics [MM] calculations, normally employed to supplement the crystallographic data, are replace by more accurate quantum mechanics [QM] calculations), combined QM/MM calculations, and accurate energy estimates to study the nature of a recent oxidised crystal structure of [NiFe] hydrogenase from Desulfovibrio fructosovorans. We show that the structure contains a mixture of several states in the active site. The experimental data is best explained by structures with a hydroxide bridge but with two of the cysteine ligands (one bridging and one terminal) partly oxidised. When the terminal Cys-543 ligand is oxidised, the sulphur occupies an alternative position, observed in several crystal structures. The Glu-25 residue, that forms a hydrogen bond to this sulphur, also changes position. A peroxide ligand may exist as a minor component in the crystal and the suggested structure is supported by the calculations. We suggest that oxidised states are slow-equilibrium mixtures of structures with a peroxide bound and structures with oxidised Cys residues, and that the former can be activated by replacement of the protonated peroxide with a H₂ or CO ligand, as has been observed in electrochemical experiments.     

Clarification of the role of protein in carbonmonoxy myoglobin by investigating electronic states

This article reports the proton tautomerization effects of distal histidine residues in carbonmonoxy myoglobin according to the density functional calculations of the whole protein. The electron eigenstates and electrostatic potential (ESP) distributed around heme and its pocket vary significantly depending on the protonation positions of the distal histidine residues. To investigate the range over which the electronic structures are affected by the proton tautomerization, the quantum mechanics/molecular mechanics (QM/MM) method is applied to probe the QM size to reproduce the atomic partial charges and ESP around the active center. Consequently, we show that these properties converged for the 300 pm QM/MM system in this study. During the analysis, we also find that amino residues such as Phe43, Val68, and Phe138 interact strongly with heme through orbital mixing, indicating that the protein is a medium not only interacting with the reaction center, but also buffering on electrons. © 2013 Wiley Periodicals, Inc.     

Flexible effective fragment QM/MM method: validation through the challenging tests

A new version of the QM/MM method, which is based on the effective fragment potential (EFP) methodology [Gordon, M. et al., J Phys Chem A 2001, 105, 293] but allows flexible fragments, is verified through calculations of model molecular systems suggested by different authors as challenging tests for QM/MM approaches. For each example, the results of QM/MM calculations for a partitioned system are compared to the results of an all-electron ab initio quantum chemical study of the entire system. In each case we were able to achieve approximately similar or better accuracy of the QM/MM results compared to those described in original publications. In all calculations we kept the same set of parameters of our QM/MM scheme. A new test example is considered when calculating the potential of internal rotation in the histidine dipeptide around the C(alpha)bond;C(beta) side chain bond.     

Systematic QM/MM investigation of factors that affect the cytochrome P450-catalyzed hydrogen abstraction of camphor

The hydrogen abstraction reaction of camphor in cytochrome P450(cam) has been investigated in the native enzyme environment by combined quantum mechanical/molecular mechanical (QM/MM) calculations and in the gas phase by density functional calculations. This work has been motivated by contradictory published QM/MM results. In an attempt to pinpoint the origin of these discrepancies, we have systematically studied the factors that may affect the computed barriers, including the QM/MM setup, the optimization procedures, and the choice of QM region, basis set, and protonation states. It is found that the ChemShell and QSite programs used in the published QM/MM calculations yield similar results at given geometries, and that the discrepancies mainly arise from two technical issues (optimization protocols and initial system preparation) that need to be well controlled in QM/MM work. In the course of these systematic investigations, new mechanistic insights have been gained. The crystallographic water 903 placed near the oxo atom of Compound I lowers the hydrogen abstraction barrier by ca. 4 kcal/mol, and thus acts as a catalyst for this reaction. Spin density may appear at the A-propionate side chain of the heme if the carboxylate group is not properly screened, which might be expected to happen during protein dynamics, but not in static equilibrium situations. There is no clear correlation between the computed A-propionate spin density and the hydrogen abstraction barrier, and hence, no support for a previously proposed side-chain mediated transition state stabilization mechanism. Standard QM/MM optimizations yield an A-propionate environment close to the X-ray structure only for protonated Asp297, and not for deprotonated Asp297, but the computed barriers are similar in both cases. An X-ray like A-propionate environment can also be obtained when deprotonated Asp297 is included in the QM region and His355 is singly protonated, but this Compound II-type species with a closed-shell porphyrin ring has a higher hydrogen abstraction barrier and should thus not be mechanistically relevant.     

Mechanism of proteolysis in matrix metalloproteinase‐2 revealed by QM/MM modeling

The mechanism of enzymatic peptide hydrolysis in matrix metalloproteinase‐2 (MMP‐2) was studied at atomic resolution through quantum mechanics/molecular mechanics (QM/MM) simulations. An all‐atom three‐dimensional molecular model was constructed on the basis of a crystal structure from the Protein Data Bank (ID: 1QIB), and the oligopeptide Ace‐Gln‐Gly～Ile‐Ala‐Gly‐Nme was considered as the substrate. Two QM/MM software packages and several computational protocols were employed to calculate QM/MM energy profiles for a four‐step mechanism involving an initial nucleophilic attack followed by hydrogen bond rearrangement, proton transfer, and C? N bond cleavage. These QM/MM calculations consistently yield rather low overall barriers for the chemical steps, in the range of 5–10 kcal/mol, for diverse QM treatments (PBE0, B3LYP, and BB1K density functionals as well as local coupled cluster treatments) and two MM force fields (CHARMM and AMBER). It, thus, seems likely that product release is the rate‐limiting step in MMP‐2 catalysis. This is supported by an exploration of various release channels through QM/MM reaction path calculations and steered molecular dynamics simulations. © 2015 Wiley Periodicals, Inc.     

The Existence of an Isolated Hydronium Ion in the Interior of Proteins

Neutron diffraction analysis studies reported an isolated hydronium ion (H₃O⁺) in the interior of d ‐xylose isomerase (XI) and phycocyanobilin‐ferredoxin oxidoreductase (PcyA). H₃O⁺ forms hydrogen bonds (H‐bonds) with two histidine side‐chains and a backbone carbonyl group in PcyA, whereas H₃O⁺ forms H‐bonds with three acidic residues in XI. Using a quantum mechanical/molecular mechanical (QM/MM) approach, we analyzed stabilization of H₃O⁺ by the protein environment. QM/MM calculations indicated that H₃O⁺ was unstable in the PcyA crystal structure, releasing a proton to an H‐bond partner His88, producing H₂O and protonated His88. On the other hand, H₃O⁺ was stable in the XI crystal structure. H‐bond partners of isolated H₃O⁺ would be practically limited to acidic residues such as aspartic and glutamic acids in the protein environment.     

A new understanding on how heme metabolism occurs in heme oxygenase: water-assisted oxo mechanism

Heme metabolism by heme oxygenase (HO) is investigated with quantum mechanical/molecular mechanical (QM/MM) calculations. A mechanism assisted by water is proposed: (1) an iron-oxo species and a water molecule are generated by the heterolytic cleavage of the O-O bond of an iron-hydroperoxo species in a similar way to P450-mediated reactions, (2) a hydrogen atom abstraction by the iron-oxo species from the generated water molecule and the C-O bond formation between the water molecule and the α-meso carbon take place simultaneously. The water molecule is hydrogen-bonded to the oxo ligand and to the water cluster in the active site of HO. The water cluster can control the position of the generated water molecule to ensure the regioselective oxidation of heme at the α-meso position, at the same time, can facilitate the oxidation by stabilizing a positive charge on the water molecule in the transition state. A key difference between HO and P450 is observed in the structure of the active site; Thr252 in P450 blocks the access of the water molecule to the α-meso position, and can thus suppress the undesired heme oxidation for P450.     

QM/MM study of mechanisms for compound I formation in the catalytic cycle of cytochrome P450cam

In the catalytic cycle of cytochrome P450cam, after molecular oxygen binds as a ligand to the heme iron atom to yield a ferrous dioxygen complex, there are fast proton transfers that lead to the formation of the active species, Compound I (Cpd I), which are not well understood because they occur so rapidly. In the present work, the conversion of the ferric hydroperoxo complex (Cpd 0) to Cpd I has been investigated by combined quantum-mechanical/molecular-mechanical (QM/MM) calculations. The residues Asp(251) and Glu(366) are considered as proton sources. In mechanism I, a proton is transported to the distal oxygen atom of the hydroperoxo group via a hydrogen bonding network to form protonated Cpd 0 (prot-Cpd0: FeOOH(2)), followed by heterolytic O-O bond cleavage that generates Cpd I and water. Although a local minimum is found for prot-Cpd0 in the Glu(366) channel, it is very high in energy (more than 20 kcal/mol above Cpd 0) and the barriers for its decay are only 3-4 kcal/mol (both toward Cpd 0 and Cpd I). In mechanism II, an initial O-O bond cleavage followed by a concomitant proton and electron transfer yields Cpd I and water. The rate-limiting step in mechanism II is O-O cleavage with a barrier of about 13-14 kcal/mol. According to the QM/MM calculations, the favored low-energy pathway to Cpd I is provided by mechanism II in the Asp(251) channel. Cpd 0 and Cpd I are of similar energies, with a slight preference for Cpd I.     

Modeling of ligation-induced helix/loop displacements in myoglobin: toward an understanding of hemoglobin allostery

Combining quantum and molecular mechanics (QM/MM) methods and protein structure prediction algorithms, helix and loop movements are computed along the pathway of CO dissociation from myoglobin (Mb). The results are compared with high-resolution crystallographic data using sequence-displacement graphs. These graphs provide an unbiased method for evaluating main-chain segmental motions; they resolve an apparent disagreement between two sets of high-resolution crystal structures for MbCO and deoxyMb. The QM/MM modeling of the CO deligation reproduces the experimentally observed spin states and photodissociated crystal structure. The principal effect of CO dissociation is shown to be a concerted rotation of the E and F helices, which hold the heme like a clamshell. The rotation is a response to deligation forces, which impel the F helix away from the heme because of the Fe spin conversion, and which allow the E helix to collapse toward the heme as nonbonded contacts on the distal side are relieved. Additional helix and loop displacements stem from these primary events. In particular, the CD loop is found to be repositioned as a result of steric interactions with the water molecule that becomes H-bonded to the distal histidine in deoxyMb. A similar EF rotation and CD loop displacement are proposed to be the first steps along the allosteric pathway from the R to the T state in hemoglobin.     

Lennard-Jones parameters for the combined QM/MM method using the B3LYP/6-31G*/AMBER potential

A combined DFT quantum mechanical and AMBER molecular mechanical potential (QM/MM) is presented for use in molecular modeling and molecular simulations of large biological systems. In our approach we evaluate Lennard-Jones parameters describing the interaction between the quantum mechanical (QM) part of a system, which is described at the B3LYP/6-31+G* level of theory, and the molecular mechanical (MM) part of the system, described by the AMBER force field. The Lennard-Jones parameters for this potential are obtained by calculating hydrogen bond energies and hydrogen bond geometries for a large set of bimolecular systems, in which one hydrogen bond monomer is described quantum mechanically and the other is treated molecular mechanically. We have investigated more than 100 different bimolecular systems, finding very good agreement between hydrogen bond energies and geometries obtained from the combined QM/MM calculations and results obtained at the QM level of theory, especially with respect to geometry. Therefore, based on the Lennard-Jones parameters obtained in our study, we anticipate that the B3LYP/6-31+G*/AMBER potential will be a precise tool to explore intermolecular interactions inside a protein environment.     

Structure of reduced and oxidized manganese superoxide dismutase: a combined computational and experimental approach

Manganese superoxide dismutases catalyze the disproportionation of the superoxide radical anion to molecular oxygen and hydrogen peroxide. Recently, atomic-resolution crystal structures of the reduced and oxidized enzymes have been reported. They show an active site with the manganese ion bound to one aspartate, three histidine residues, and a solvent molecule. In this paper, we combine crystallographic refinement with quantum mechanical methods to show that the solvent ligand is undoubtedly a water molecule in the reduced state. However, the putative oxidized structure is to a large extent reduced during data collection, so that it contains a mixture of the Mn2+ and Mn3+ structure. The crystal structures show that the Mn-bound solvent molecule accepts a hydrogen bond from the side chain of the conserved Gln-146 residue. If the solvent ligand is water, then this could lead to a steric clash, but it is avoided by the plane of water molecule forming an angle of 72 degrees to the Mn-O bond. Such a conformation is also found outside the enzyme, giving a minimal destabilization of the reduced state. We show by molecular dynamics simulations that the suggested Mn2+-H2O and Mn3+-OH- structures are stable. Moreover, we show that the superoxide substrate may bind both in the first coordination sphere of the Mn ion, opposite to the aspartate ligand, or in the second sphere, close to the conserved Tyr-34 and His-30 residues, approximately 5 A from Mn. However, the second-sphere structures are not stable in long molecular dynamics simulations. We see no difference in the coordination between the reduced and the oxidized states of the enzyme.     

Computational characterization of reaction intermediates in the photocycle of the sensory domain of the AppA blue light photoreceptor

The AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark-adapted and light-induced functional states. We characterized possible reaction intermediates in the photocycle of AppA BLUF. Molecular dynamics (MD), quantum chemical and quantum mechanical-molecular mechanical (QM/MM) calculations were carried out to describe several stable structures of a molecular system modeling the protein. The coordinates of heavy atoms from the crystal structure (PDB code 2IYG) of the protein in the dark state served as starting point for 10 ns MD simulations. Representative MD frames were used in QM(B3LYP/cc-pVDZ)/MM(AMBER) calculations to locate minimum energy configurations of the model system. Vertical electronic excitation energies were estimated for the molecular clusters comprising the quantum subsystems of the QM/MM optimized structures using the SOS-CIS(D) quantum chemistry method. Computational results support the occurrence of photoreaction intermediates that are characterized by spectral absorption bands between those of the dark and light states. They agree with crystal structures of reaction intermediates (PDB code 2IYI) observed in the AppA BLUF domain. Transformations of the Gln63 side chain stimulated by photo-excitation and performed with the assistance of the chromophore and the Met106 side chain are responsible for these intermediates.     

Phosphorylation reaction in cAPK protein kinase-free energy quantum mechanical/molecular mechanics simulations

We present results of a theoretical analysis of the phosphorylation reaction in cAMP-dependent protein kinase using a combined quantum mechanical and molecular mechanics (QM/MM) approach. Detailed analysis of the reaction pathway is provided using a novel QM/MM implementation of the nudged elastic band method, finite temperature fluctuations of the protein environment are taken into account using free energy calculations, and an analysis of hydrogen bond interactions is performed on the basis of calculated frequency shifts. The late transfer of the substrate proton to the conserved aspartate (D166), the activation free energy of 15 kcal/mol, and the slight exothermic (-3 kcal/mol) character of the reaction are all consistent with the experimental data. The near attack conformation of D166 in the reactant state is maintained by interactions with threonine-201, asparagine-177, and most notably by a conserved water molecule serving as a strong structural link between the primary metal ion and the D166. The secondary Mg ion acts as a Lewis acid, attacking the beta-gamma bridging oxygen of ATP. This interaction, along with a strong hydrogen bond between the D166 and the substrate, contributes to the stabilization of the transition state. Lys-168 maintains a hydrogen bond to a transferring phosphoryl group throughout a reaction process. This interaction increases in the product state and contributes to its stabilization.     

QM/MM modeling of compound I active species in cytochrome P450, cytochrome C peroxidase, and ascorbate peroxidase

QM/MM calculations provide a means for predicting the electronic structure of the metal center in metalloproteins. Two heme peroxidases, Cytochrome c Peroxidase (CcP) and Ascorbate Peroxidase (APX), have a structurally very similar active site, yet have active intermediates with very different electronic structures. We review our recent QM/MM calculations on these systems, and present new computational data. Our results are in good agreement with experiment, and suggest that the difference in electronic structure is due to a large number of small differences in structure from one protein to another. We also discuss recent QM/MM calculations on the active species of cytochrome P450, in which a similar sensitivity of the electronic structure to the environment is found. However, this does not appear to explain different catalytic profiles of the different drug-metabolizing isoforms of this class of enzyme.     

Mechanism of C-terminal intein cleavage in protein splicing from QM/MM molecular dynamics simulations

Protein splicing is a post-translational process in which a biologically inactive protein is activated by the release of a segment denoted as an intein. The process involves four steps. In the third, the scission of the intein takes place after the cyclization of the last amino acid of the segment, an asparagine. Little is known about the chemical reaction necessary for this cyclization. Experiments demonstrate that two histidines (the penultimate amino acid of the intein, and a histidine located 10 amino acids upstream) are relevant in the cyclization of the asparagine. We have investigated the mechanism and determinants of reaction in the GyrA intein focusing on the requirements for asparagine activation for its cyclization. First, the influence that the protonation states of these two histidines have on the orientation of the asparagine side chain is investigated by means of molecular dynamics simulation. Molecular dynamics simulations using the CHARMM27 force field were carried out on the three possible protonation states for each of these two histidines. The results indicate that the only protonation state in which the conformation of the system is suitable for cyclization is when the penultimate histidine is fully protonated (positively charged), and the upstream histidine is in the His(ε) neutral tautomeric form. The free energy profile for the reaction in which the asparagine is activated by a proton transfer to the upstream histidine is presented, computed by hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics at the SCCDFTB/CHARMM27 level of theory. The calculated free energy barrier for the reaction is 19.0 kcal mol(-1). B3LYP/6-31+G(d) QM/MM single-point calculations give a qualitatively a similar energy profile, although with somewhat higher energy barriers, in good agreement with the value derived from experiment of 25 kcal mol(-1) at 60 °C. QM/MM molecular dynamics simulations of the reactant, activated reactant and intermediate states highlight the importance of the Arg181-Val182-Asp183 segment in catalysing the reaction. Overall, the results indicate that nucleophilic activation of the asparagine for its cyclization by the upstream histidine acting as the base is a plausible mechanism for the C-terminal cleavage in protein splicing.     

The elusive oxidant species of cytochrome P450 enzymes: characterization by combined quantum mechanical/molecular mechanical (QM/MM) calculations

The primary oxidant of cytochrome P450 enzymes, Compound I, is hard to detect experimentally; in the case of cytochrome P450(cam), this intermediate does not accumulate in solution during the catalytic cycle even at temperatures as low as 200 K (ref 4). Theory can play an important role in characterizing such elusive species. We present here combined quantum mechanical/molecular mechanical (QM/MM) calculations of Compound I of cytochrome P450(cam) in the full enzyme environment as well as density functional studies of the isolated QM region. The calculations assign the ground state of the species, quantify the effect of polarization and hydrogen bonding on its properties, and show that the protein environment and its specific hydrogen bonding to the cysteinate ligand are crucial for sustaining the Fe-S bond and for preventing the full oxidation of the sulfur.     

Quantum capping potentials with point charges: a simple QM/MM approach for the calculation of large-molecule NMR shielding tensors

A simple quantum-mechanics/molecular-mechanics (QM/MM) approach for calculating NMR shielding tensors (sigma) is presented. The method involves capping the QM region with quantum capping potentials (QCPs) and representing the MM region with point charges. Test calculations on simple systems without MM charges show that calculated sigma values improve relative to the full QM results with increasing distance between the capped bond and chromophore. Calculations on the histidine amino acid and cytosine monophosphate (CMP) nucleic acid show that the use of QCPs with point charges result in mean errors in the isotropic component of sigma that are less than 1.6 ppm. The results also reveal that, contrary to previous work, the explicit effect of point charges on sigma through coupling with gauge factors, as in the gauge including atomic orbital approach, is minimal for the CMP molecule. The present QM/MM approach for calculating sigma is easy to apply and requires no code modification.     

On the origin of the stabilization of the zwitterionic resting state of cysteine proteases: a theoretical study

Papain-like cysteine proteases are ubiquitous proteolytic enzymes. The protonated His199/deprotonated Cys29 ion pair (cathepsin B numbering) in the active site is essential for their proper functioning. The presence of this ion pair stands in contrast to the corresponding intrinsic residue p K a values, indicating a strong influence of the enzyme environment. In the present work we show by molecular dynamics simulations on quantum mechanical/molecular mechanical (QM/MM) potentials that the ion pair is stabilized by a complex hydrogen bond network which comprises several amino acids situated in the active site of the enzyme and 2-4 water molecules. QM/MM reaction path computations for the proton transfer from His199 to the thiolate of the Cys29 moiety indicate that the ion pair is about 32-36 kJ mol (-1) more stable than the neutral form if the whole hydrogen bonding network is active. Without any hydrogen bonding network the ion pair is predicted to be significantly less stable than the neutral form. QM/MM charge deletion analysis and QM model calculations are used to quantify the stabilizing effect of the active-site residues and the L1 helix in favor of the zwitterionic form. The active-site water molecules contribute about 30 kJ mol (-1) to the overall stabilization. Disruption of the hydrogen bonding network upon substrate binding is expected to enhance the nucleophilic reactivity of the thiolate.