Interplay of halogen bonding and solvation in protein-ligand binding.

Halogen bonding is increasingly utilized in efforts to achieve high affinity and selectivity of molecules designed to bind proteins, making it paramount to understand the relationship between structure, dynamics, and thermodynamic driving forces. We present a detailed analysis addressing this problem using a series of protein-ligand complexes involving single halogen substitutions - F, Cl, Br, and I - and nearly identical structures. Isothermal titration calorimetry reveals an increasingly favorable binding enthalpy from F to I that correlates with the halogen size and σ-hole electropositive character, but is partially counteracted by unfavorable entropy, which is constant from F to Cl and Br, but worse for I. Consequently, the binding free energy is roughly equal for Cl, Br, and I. QM and solvation-free-energy calculations reflect an intricate balance between halogen bonding, hydrogen bonds, and solvation. These advances have the potential to aid future drug design initiatives involving halogenated compounds.

CO Oxidation Mechanism of Silver-Substituted Mo/Cu CO-Dehydrogenase - Analogies and Differences to the Native Enzyme.

The aerobic oxidation of carbon monoxide to carbon dioxide is catalysed by the Mo/Cu-containing CO-dehydrogenase enzyme in the soil bacterium Oligotropha carboxidovorans, enabling the organism to grow on the small gas molecule as carbon and energy source. It was shown experimentally that silver can be substituted for copper in the active site of Mo/Cu CODH to yield a functional enzyme. In this study, we employed QM/MM calculations to investigate whether the reaction mechanism of the silver-substituted enzyme is similar to that of the native enzyme. Our results suggest that the Ag-substituted enzyme can oxidize CO and release CO2 following the same reaction steps as the native enzyme, with a computed rate-limiting step of 10.4 kcal/mol, consistent with experimental findings. Surprisingly, lower activation energies for C-O bond formation have been found in the presence of silver. Furthermore, comparison of rate constants for reduction of copper- and silver-containing enzymes suggests a discrepancy in the transition state stabilization upon silver substitution. We also evaluated the effects that differences in the water-active site interaction may exert on the overall energy profile of catalysis. Finally, the formation of a thiocarbonate intermediate along the catalytic pathway was found to be energetically unfavorable for the Ag-substituted enzyme. This finding aligns with the hypothesis proposed for the wild-type form, suggesting that the creation of such species may not be necessary for the enzymatic catalysis of CO oxidation.

Scalar Relativistic All-Electron and Pseudopotential Ab Initio Study of a Minimal Nitrogenase [Fe(SH)4H]- Model Employing Coupled-Cluster and Auxiliary-Field Quantum Monte Carlo Many-Body Methods.

Nitrogenase is the only enzyme that can cleave the triple bond in N2, making nitrogen available to organisms. The detailed mechanism of this enzyme is currently not known, and computational studies are complicated by the fact that different density functional theory (DFT) methods give very different energetic results for calculations involving nitrogenase models. Recently, we designed a [Fe(SH)4H]- model with the fifth proton binding either to Fe or S to mimic different possible protonation states of the nitrogenase active site. We showed that the energy difference between these two isomers (ΔE) is hard to estimate with quantum-mechanical methods. Based on nonrelativistic single-reference coupled-cluster (CC) calculations, we estimated that the ΔE is 101 kJ/mol. In this study, we demonstrate that scalar relativistic effects play an important role and significantly affect ΔE. Our best revised single-reference CC estimates for ΔE are 85-91 kJ/mol, including energy corrections to account for contributions beyond triples, core-valence correlation, and basis-set incompleteness error. Among coupled-cluster approaches with approximate triples, the canonical CCSD(T) exhibits the largest error for this problem. Complementary to CC, we also used phaseless auxiliary-field quantum Monte Carlo calculations (ph-AFQMC). We show that with a Hartree-Fock (HF) trial wave function, ph-AFQMC reproduces the CC results within 5 ± 1 kJ/mol. With multi-Slater-determinant (MSD) trials, the results are 82-84 ± 2 kJ/mol, indicating that multireference effects may be rather modest. Among the DFT methods tested, τ-HCTH, r2SCAN with 10-13% HF exchange with and without dispersion, and O3LYP/O3LYP-D4, and B3LYP*/B3LYP*-D4 generally perform the best. The r2SCAN12 (with 12% HF exchange) functional mimics both the best reference MSD ph-AFQMC and CC ΔE results within 2 kJ/mol.

QM/MM study of the catalytic reaction of aphid myrosinase.

Brevicoryne brassicae, an aphid species, exclusively consumes plants from the Brassicaceae family and employs a sophisticated defense mechanism involving a myrosinase enzyme that breaks down glucosinolates obtained from its host plants. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations to study the catalytic reaction of aphid myrosinase. A proper QM region to study the myrosinase reaction should contain the whole substrate, models of Gln-19, His-122, Asp-124, Asn-166, Glu-167, Lys-173, Tyr-180, Val-228, Tyr-309, Tyr-346, Ile-347, Glu-374, Glu-423, Trp-424, and a water molecule. The calculations show that Asp-124 and Glu-423 must be charged, His-122 must be protonated on NE2, and Glu-167 must be protonated on OE2. Our model reproduces the anomeric retaining characteristic of myrosinase and indicates that the deglycosylation reaction is the rate-determining step of the reaction. Based on the calculations, we propose a reaction mechanism for aphid myrosinase-mediated hydrolysis of glucosinolates with an overall barrier of 15.2 kcal/mol. According to the results, removing a proton from Arg-312 or altering it to valine or methionine increases glycosylation barriers but decreases the deglycosylation barrier.

H2 formation from the E2-E4 states of nitrogenase.

Nitrogenase is the only enzyme that can cleave the strong triple bond in N2, making nitrogen available for biological lifeforms. The active site is a MoFe7S9C cluster (the FeMo cluster) that binds eight electrons and protons during one catalytic cycle, giving rise to eight intermediate states E0-E7. It is experimentally known that N2 binds to the E4 state and that H2 is a compulsory byproduct of the reaction. However, formation of H2 is also an unproductive side reaction that should be avoided, especially in the early steps of the reaction mechanism (E2 and E3). Here, we study the formation of H2 for various structural interpretations of the E2-E4 states using combined quantum mechanical and molecular mechanical (QM/MM) calculations and four different density-functional theory methods. We find large differences in the predictions of the different methods. B3LYP strongly favours protonation of the central carbide ion and H2 cannot form from such structures. On the other hand, with TPSS, r2SCAN and TPSSh, H2 formation is strongly exothermic for all structures and En and therefore need strict kinetic control to be avoided. For the E2 state, the kinetic barriers for the low-energy structures are high enough to avoid H2 formation. However, for both the E3 and E4 states, all three methods predict that the best structure has two hydride ions bridging the same pair of Fe ions (Fe2 and Fe6) and these two ions can combine to form H2 with an activation barrier of only 29-57 kJ mol-1, corresponding to rates of 7 × 102 to 5 × 107 s-1, i.e. much faster than the turnover rate of the enzyme (1-5 s-1). We have also studied H-atom movements within the FeMo cluster, showing that the various protonation states can quite freely be interconverted (activation barriers of 12-69 kJ mol-1).

Multireference Protonation Energetics of a Dimeric Model of Nitrogenase Iron-Sulfur Clusters.

Characterizing the electronic structure of the iron-sulfur clusters in nitrogenase is necessary to understand their role in the nitrogen fixation process. One challenging task is to determine the protonation state of the intermediates in the nitrogen fixing cycle. Here, we use a dimeric iron-sulfur model to study relative energies of protonation at C, S, or Fe. Using a composite method based on coupled cluster and density matrix renormalization group energetics, we converge the relative energies of four protonated configurations with respect to basis set and correlation level. We find that accurate relative energies require large basis sets as well as a proper treatment of multireference and relativistic effects. We have also tested ten density functional approximations for these systems. Most of them give large errors in their relative energies. The best performing functional in this system is B3LYP, which gives mean absolute and maximum deviations of only 10 and 13 kJ/mol with respect to our correlated wave function estimates, respectively, comparable to the uncertainty in our correlated estimates. Our work provides benchmark results for the calibration of new approximate electronic structure methods and density functionals for these problems.

Protonation of Homocitrate and the E1 State of Fe-Nitrogenase Studied by QM/MM Calculations.

Nitrogenase is the only enzyme that can cleave the strong triple bond in N2, making nitrogen available for biological life. There are three isozymes of nitrogenase, differing in the composition of the active site, viz., Mo, V, and Fe-nitrogenase. Recently, the first crystal structure of Fe-nitrogenase was presented. We have performed the first combined quantum mechanical and molecular mechanical (QM/MM) study of Fe-nitrogenase. We show with QM/MM and quantum-refinement calculations that the homocitrate ligand is most likely protonated on the alcohol oxygen in the resting E0 state. The most stable broken-symmetry (BS) states are the same as for Mo-nitrogenase, i.e., the three Noodleman BS7-type states (with a surplus of ß spin on the eighth Fe ion), which maximize the number of nearby antiferromagnetically coupled Fe-Fe pairs. For the E1 state, we find that protonation of the S2B µ2 belt sulfide ion is most favorable, 14-117 kJ/mol more stable than structures with a Fe-bound hydride ion (the best has a hydride ion on the Fe2 ion) calculated with four different density-functional theory methods. This is similar to what was found for Mo-nitrogenase, but it does not explain the recent EPR observation that the E1 state of Fe-nitrogenase should contain a photolyzable hydride ion. For the E1 state, many BS states are close in energy, and the preferred BS state differs depending on the position of the extra proton and which density functional is used.

Assessment of DFT functionals for a minimal nitrogenase [Fe(SH)4H]- model employing state-of-the-art ab initio methods.

We have designed a [Fe(SH)4H]- model with the fifth proton binding either to Fe or S. We show that the energy difference between these two isomers (∆E) is hard to estimate with quantum-mechanical (QM) methods. For example, different density functional theory (DFT) methods give ∆E estimates that vary by almost 140 kJ/mol, mainly depending on the amount of exact Hartree-Fock included (0%-54%). The model is so small that it can be treated by many high-level QM methods, including coupled-cluster (CC) and multiconfigurational perturbation theory approaches. With extrapolated CC series (up to fully connected coupled-cluster calculations with singles, doubles, and triples) and semistochastic heat-bath configuration interaction methods, we obtain results that seem to be converged to full configuration interaction results within 5 kJ/mol. Our best result for ∆E is 101 kJ/mol. With this reference, we show that M06 and B3LYP-D3 give the best results among 35 DFT methods tested for this system. Brueckner doubles coupled cluster with perturbaitve triples seems to be the most accurate coupled-cluster approach with approximate triples. CCSD(T) with Kohn-Sham orbitals gives results within 4-11 kJ/mol of the extrapolated CC results, depending on the DFT method. Single-reference CC calculations seem to be reasonably accurate (giving an error of ∼5 kJ/mol compared to multireference methods), even if the D1 diagnostic is quite high (0.25) for one of the two isomers.

Two local minima for structures of [4Fe-4S] clusters obtained with density functional theory methods.

[4Fe-4S] clusters are essential cofactors in many proteins involved in biological redox-active processes. Density functional theory (DFT) methods are widely used to study these clusters. Previous investigations have indicated that there exist two local minima for these clusters in proteins. We perform a detailed study of these minima in five proteins and two oxidation states, using combined quantum mechanical and molecular mechanical (QM/MM) methods. We show that one local minimum (L state) has longer Fe-Fe distances than the other (S state), and that the L state is more stable for all cases studied. We also show that some DFT methods may only obtain the L state, while others may obtain both states. Our work provides new insights into the structural diversity and stability of [4Fe-4S] clusters in proteins, and highlights the importance of reliable DFT methods and geometry optimization. We recommend r2SCAN for optimizing [4Fe-4S] clusters in proteins, which gives the most accurate structures for the five proteins studied.

N2 binding to the E0-E4 states of nitrogenase.

Nitrogenase is the only enzyme that can convert N2 into NH3. The reaction requires the addition of eight electrons and protons to the enzyme and the mechanism is normally described by nine states, E0-E8, differing in the number of added electrons. Experimentally, it is known that three or four electrons need to be added before the enzyme can bind N2. We have used combined quantum mechanical and molecular mechanics methods to study the binding of N2 to the E0-E4 states of nitrogenase, using four different density functional theory (DFT) methods. We test many different structures for the E2-E4 states and study binding both to the Fe2 and Fe6 ions of the active-site FeMo cluster. Unfortunately, the results depend quite strongly on the DFT methods. The TPSS method gives the strongest bonding and prefers N2 binding to Fe6. It is the only method that reproduces the experimental observation of unfavourable binding to the E0-E2 states and favourable binding to E3 and E4. The other three methods give weaker binding, preferably to Fe2. B3LYP strongly favours structures with the central carbide ion triply protonated. The other three methods suggest that states with the S2B ligand dissociated from either Fe2 or Fe6 are competitive for the E2-E4 states. Moreover, such structures with two hydride ions both bridging Fe2 and Fe6 are the best models for E4 and also for the N2-bound E3 and E4 states. However, for E4, other structures are often close in energy, e.g. structures with one of the hydride ions bridging instead Fe3 and Fe7. Finally, we find no support for the suggestion that reductive elimination of H2 from the two bridging hydride ions in the E4 state would enhance the binding of N2.

Comparison of force fields to study the zinc-finger containing protein NPL4, a target for disulfiram in cancer therapy.

Molecular dynamics (MD) simulations are a powerful approach to studying the structure and dynamics of proteins related to health and disease. Advances in the MD field allow modeling proteins with high accuracy. However, modeling metal ions and their interactions with proteins is still challenging. NPL4 is a zinc-binding protein and works as a cofactor for p97 to regulate protein homeostasis. NPL4 is of biomedical importance and has been proposed as the target of disulfiram, a drug recently repurposed for cancer treatment. Experimental studies proposed that the disulfiram metabolites, bis-(diethyldithiocarbamate)­copper and cupric ions, induce NPL4 misfolding and aggregation. However, the molecular details of their interactions with NPL4 and consequent structural effects are still elusive. Here, biomolecular simulations can help to shed light on the related structural details. To apply MD simulations to NPL4 and its interaction with copper the first important step is identifying a suitable force field to describe the protein in its zinc-bound states. We examined different sets of non-bonded parameters because we want to study the misfolding mechanism and cannot rule out that the zinc may detach from the protein during the process and copper replaces it. We investigated the force-field ability to model the coordination geometry of the metal ions by comparing the results from MD simulations with optimized geometries from quantum mechanics (QM) calculations using model systems of NPL4. Furthermore, we investigated the performance of a force field including bonded parameters to treat copper ions in NPL4 that we obtained based on QM calculations.

Catalytic Reaction Mechanism of Glyoxalase II: A Quantum Mechanics/Molecular Mechanics Study.

Methylglyoxal (MG) is a reactive and toxic compound produced in carbohydrate, lipid, and amino acid metabolism. The glyoxalase system is the main detoxifying route for MG and consists of two enzymes, glyoxalase I (GlxI) and glyoxalase II (GlxII). GlxI catalyzes the formation of S-d-lactoylglutathione from hemithioacetal, and GlxII converts this intermediate to d-lactate. A relationship between the glyoxalase system and some diseases like diabetes has been shown, and inhibiting enzymes of this system may be an effective means of controlling certain diseases. A detailed understanding of the reaction mechanism of an enzyme is essential to the rational design of competitive inhibitors. In this work, we use quantum mechanics/molecular mechanics (QM/MM) calculations and energy refinement utilizing the big-QM and QM/MM thermodynamic cycle perturbation methods to propose a mechanism for the GlxII reaction that starts with a nucleophilic attack of the bridging OH- group on the substrate. The coordination of the substrate to the Zn ions places its electrophilic center close to the hydroxide group, enabling the reaction to proceed. Our estimated reaction energies are in excellent agreement with experimental data, thus demonstrating the reliability of our approach and the proposed mechanism. Additionally, we examined alternative protonation states of Asp-29, Asp-58, Asp-134, and the bridging hydroxide ion in the catalytic process. However, these give less favorable reactions, a poorer reproduction of the crystal structure geometry of the active site, and higher root-mean-squared deviations of the active site residues in molecular dynamics simulations.

A computational study of the reaction mechanism and stereospecificity of dihydropyrimidinase.

Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of ß-amino acids. It catalyses the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to the corresponding N-carbamoyl-ß-amino acids. This enzyme has the potential to be used as a tool in the production of ß-amino acids. Here, the reaction mechanism and origin of stereospecificity of DHPases from Saccharomyces kluyveri and Sinorhizobium meliloti CECT4114 were investigated and compared using a quantum mechanical cluster approach based on density functional theory. Two models of the enzyme active site were designed from the X-ray crystal structure of the native enzyme: a small cluster to characterize the mechanism and the stationary points and a large model to probe the stereospecificity and the role of stereo-gate-loop (SGL) residues. It is shown that a hydroxide ion first performs a nucleophilic attack on the substrate, followed by the abstraction of a proton by Asp358, which occurs concertedly with protonation of the ring nitrogen by the same residue. For the DHT substrate, the enzyme displays a preference for the L-configuration, in good agreement with experimental observation. Comparison of the reaction energetics of the two models reveals the importance of SGL residues in the stereospecificity of catalysis. The role of the conserved Tyr172 residue in transition-state stabilization is confirmed as the Tyr172Phe mutation increases the activation barrier of the reaction by ∼8 kcal mol-1. A detailed understanding of the catalytic mechanism of the enzyme could offer insight for engineering in order to enhance its activity and substrate scope.

Histidine oxidation in lytic polysaccharide monooxygenase.

The lytic polysaccharide monooxygenases (LPMOs) comprise a super-family of copper enzymes that boost the depolymerisation of polysaccharides by oxidatively disrupting the glycosidic bonds connecting the sugar units. Industrial use of LPMOs for cellulose depolymerisation has already begun but is still far from reaching its full potential. One issue is that the LPMOs self-oxidise and thereby deactivate. The mechanism of this self-oxidation is unknown, but histidine residues coordinating to the copper atom are the most susceptible. An unusual methyl modification of the NE2 atom in one of the coordinating histidine residues has been proposed to have a protective role. Furthermore, substrate binding is also known to reduce oxidative damage. We here for the first time investigate the mechanism of histidine oxidation with combined quantum and molecular mechanical (QM/MM) calculations, with outset in intermediates previously shown to form from a reaction with peroxide and a reduced LPMO. We show that an intermediate with a [Cu-O]+ moiety is sufficiently potent to oxidise the nearest C-H bond on both histidine residues, but methylation of the NE2 atom of His-1 increases the reaction barrier of this reaction. The substrate further increases the activation barrier. We also investigate a [Cu-OH]2+ intermediate with a deprotonated tyrosine radical. This intermediate was previously proposed to have a protective role, and we also find it to have higher barriers than the corresponding a [Cu-O]+ intermediate.

How general is the effect of the bulkiness of organic ligands on the basicity of metal-organic catalysts? H2-evolving Mo oxides/sulphides as case studies.

Tailoring the activity of an organometallic catalyst usually requires a targeted ligand design. Tuning the ligand bulkiness and tuning the electronic properties are popular approaches, which are somehow interdependent because substituents of different sizes within ligands can determine inter alia the occurrence of different degrees of inductive effects. Ligand basicity, in particular, turned out to be a key property for the modulation of protonation reactions occurring in vacuo at the metals in complexes bearing organophosphorus ligands; however, when the same reactions take place in a polar organic solvent, their energetics becomes dependent on the trade-off between ligand basicity and bulkiness, with the polarity of the solvent playing a key role in this regard [Bancroft et al., Inorg. Chem., 1986, 25, 3675; Rovaletti et al., J. Phys. Org. Chem., 2018, 31, e3748]. In the present contribution, we carried out molecular dynamics and density functional theory calculations on water-soluble Mo-based catalysts for proton reduction, in order to study the energetics of protonation reactions in complexes where the incipient proton binds a catalytically active ligand (i.e., an oxide or a disulphide). We considered complexes either soaked in water or in a vacuum, and featuring N-based ancillary ligands of different bulkiness (i.e. cages constituted either by pyridine or isoquinoline moieties). Our results show that the energetics of protonation events can be affected by ancillary ligand bulkiness even when the metal center does not play the role of the H+ acceptor. In vacuo, protonation at the O or S atom in the α position relative to the metal in complexes featuring the bulky isoquinoline-based ligand is more favored by around 10 kcal mol-1 when compared to the case of the pyridine-based counterparts, a difference that is almost zero when the same reactions occur in water. Such an outcome is rationalized in light of the different electrostatic properties of complexes bearing ancillary ligands of different sizes. The overall picture from theory indicates that such effects of ligand bulkiness can be relevant for the design of green chemistry catalysts that undergo protonation steps in water solutions.

QM/MM Study of Partial Dissociation of S2B for the E2 Intermediate of Nitrogenase.

Nitrogenase is the only enzyme that can cleave the triple bond in N2, making nitrogen available for all lifeforms. Previous computational studies have given widely diverging results regarding the reaction mechanism of the enzyme. For example, some recent studies have suggested that one of the µ2-bridging sulfide ligands (S2B) may dissociate from one of the Fe ions when protonated in the doubly reduced and protonated E2 state, whereas other studies indicated that such half-dissociated states are unfavorable. We have examined how the relative energies of 26 structures of the E2 state depend on details of combined quantum mechanical and molecular mechanical (QM/MM) calculations. We show that the selection of the broken-symmetry state, the basis set, relativistic effects, the size of the QM system, relaxation of the surroundings, and the conformations of the bound protons may affect the relative energies of the various structures by up to 12, 22, 9, 20, 37, and 33 kJ/mol, respectively. However, they do not change the preferred type of structures. On the other hand, the choice of the DFT functional strongly affects the preferences. The hybrid B3LYP functional strongly prefers doubly protonation of the central carbide ion, but such a structure is not consistent with experimental EPR data. Other functionals suggest structures with a hydride ion, in agreement with the experiments, and show that the ion bridges between Fe2 and Fe6. Moreover, there are two structures of the same type that are degenerate within 1-5 kJ/mol, in agreement with the observation of two EPR signals. However, the pure generalized gradient approximation (GGA) functional TPSS favors structures with a protonated S2B also bridging Fe2 and Fe6, whereas r2SCAN (meta-GGA) and TPSSh (hybrid) prefer structures with S2B dissociated from Fe2 (but remaining bound to Fe6). The energy difference between the two types of structure is so small (7-18 kJ/mol) that both types need to be considered in future investigations of the mechanism of nitrogenase.

Proton Transfer Pathways in Nitrogenase with and without Dissociated S2B.

Nitrogenase is the only enzyme that can convert N2 to NH3 . Crystallographic structures have indicated that one of the sulfide ligands of the active-site FeMo cluster, S2B, can be replaced by an inhibitor, like CO and OH- , and it has been suggested that it may be displaced also during the normal reaction. We have investigated possible proton transfer pathways within the FeMo cluster during the conversion of N2 H2 to two molecules of NH3 , assuming that the protons enter the cluster at the S3B, S4B or S5A sulfide ions and are then transferred to the substrate. We use combined quantum mechanical and molecular mechanical (QM/MM) calculations with the TPSS and B3LYP functionals. The calculations indicate that the barriers for these reactions are reasonable if the S2B ligand remains bound to the cluster, but they become prohibitively high if S2B has dissociated. This suggests that it is unlikely that S2B reversibly dissociates during the normal reaction cycle.

Comparison of Grand Canonical and Conventional Molecular Dynamics Simulation Methods for Protein-Bound Water Networks.

Water molecules play important roles in all biochemical processes. Therefore, it is of key importance to obtain information of the structure, dynamics, and thermodynamics of water molecules around proteins. Numerous computational methods have been suggested with this aim. In this study, we compare the performance of conventional and grand-canonical Monte Carlo (GCMC) molecular dynamics (MD) simulations to sample the water structure, as well GCMC and grid-based inhomogeneous solvation theory (GIST) to describe the energetics of the water network. They are evaluated on two proteins: the buried ligand-binding site of a ferritin dimer and the solvent-exposed binding site of galectin-3. We show that GCMC/MD simulations significantly speed up the sampling and equilibration of water molecules in the buried binding site, thereby making the results more similar for simulations started from different states. Both GCMC/MD and conventional MD reproduce crystal-water molecules reasonably for the buried binding site. GIST analyses are normally based on restrained MD simulations. This improves the precision of the calculated energies, but the restraints also significantly affect both absolute and relative energies. Solvation free energies for individual water molecules calculated with and without restraints show a good correlation, but with large quantitative differences. Finally, we note that the solvation free energies calculated with GIST are ∼5 times larger than those estimated by GCMC owing to differences in the reference state.

Computationally enhanced X-ray diffraction analysis of a gold(III) complex interacting with the human telomeric DNA G-quadruplex. Unravelling non-unique ligand positioning.

The crystal structure of the human telomeric DNA Tel24 G-quadruplex (Tel24 = TAG3(T2AG3)3T) in complex with the novel [AuL] species (with L = 2,4,6-tris(2-pyrimidyl)-1,3,5-triazine - TPymT-α) was solved by a novel joint molecular mechanical (MM)/quantum mechanical (QM) innovative approach. The quantum-refinement crystallographic method (crystallographic refinement enhanced with quantum mechanical calculation) was adapted to treat the [AuL]/G-quadruplex structure, where each gold complex in the binding site was found spread over four equally occupied positions. The four positions were first determined by docking restrained to the crystallographically determined metal ions' coordinates. Then, the quantum refinement method was used to resolve the poorly defined density around the ligands and improve the crystallographic determination, revealing that the binding preferences of this metallodrug toward Tel24 G-quadruplex arise from a combined effect of pyrimidine stacking, metal-guanine interactions and charge-charge neutralizing action of the π-acid triazine. The occurrence of interaction in solution with the Tel24 G-quadruplex DNA was further proved through DNA melting experiments, which showed a slight destabilisation of the quadruplex upon adduct formation.

Benchmark Study of Redox Potential Calculations for Iron-Sulfur Clusters in Proteins.

Redox potentials have been calculated for 12 different iron-sulfur sites of 6 different types with 1-4 iron ions. Structures were optimized with combined quantum mechanical and molecular mechanical (QM/MM) methods, and the redox potentials were calculated using the QM/MM energies, single-point QM methods in a continuum solvent or by QM/MM thermodynamic cycle perturbations. We show that the best results are obtained with a large QM system (∼300 atoms, but a smaller QM system, ∼150 atoms, can be used for the QM/MM geometry optimization) and a large value of the dielectric constant (80). For absolute redox potentials, the B3LYP density functional method gives better results than TPSS, and the results are improved with a larger basis set. However, for relative redox potentials, the opposite is true. The results are insensitive to the force field (charges of the surroundings) used for the QM/MM calculations or whether the protein and solvent outside the QM system are relaxed or kept fixed at the crystal structure. With the best approach for relative potentials, mean absolute and maximum deviations of 0.17 and 0.44 V, respectively, are obtained after removing a systematic error of -0.55 V. Such an approach can be used to identify the correct oxidation states involved in a certain redox reaction.