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.

Large barrier behavior of the rate constant from the diffusion equation.

Many processes in chemistry, physics, and biology depend on thermally activated events in which the system changes its state by surmounting an activation barrier. Examples range from chemical reactions to protein folding and nucleation events. Parameterized forms of the mean field potential are often employed in the stochastic modeling of activated processes. In this contribution, we explore the alternative of employing parameterized forms of the equilibrium distribution by means of symmetric linear combination of two Gaussian functions. Such a procedure leads to flexible and convenient models for the landscape and the energy barrier whose features are controlled by the second moments of these Gaussian functions. The rate constants are examined through the solution of the corresponding diffusion problem, that is, the Fokker-Planck-Smoluchowski equation specified according to the parameterized equilibrium distribution. Numerical calculations clearly show that the asymptotic limit of large barriers does not agree with the results of the Kramers theory. The underlying reason is that the linear scaling of the potential, the procedure justifying the Kramers theory, cannot be applied when dealing with parameterized forms of the equilibrium distribution. A different kind of asymptotic analysis is then required and we introduce the appropriate theory when the equilibrium distribution is represented as a symmetric linear combination of two Gaussian functions: first in the one-dimensional case and afterward in the multidimensional diffusion model.

Evaluation of docking procedures reliability in affitins-partners interactions.

Affitins constitute a class of small proteins belonging to Sul7d family, which, in microorganisms such as Sulfolobus acidocaldarius, bind DNA preventing its denaturation. Thanks to their stability and small size (60-66 residues in length) they have been considered as ideal candidates for engineering and have been used for more than 10 years now, for different applications. The individuation of a mutant able to recognize a specific target does not imply the knowledge of the binding geometry between the two proteins. However, its identification is of undoubted importance but not always experimentally accessible. For this reason, computational approaches such as protein-protein docking can be helpful for an initial structural characterization of the complex. This method, which produces tens of putative binding geometries ordered according to a binding score, needs to be followed by a further reranking procedure for finding the most plausible one. In the present paper, we use the server ClusPro for generating docking models of affitins with different protein partners whose experimental structures are available in the Protein Data Bank. Then, we apply two protocols for reranking the docking models. The first one investigates their stability by means of Molecular Dynamics simulations; the second one, instead, compares the docking models with the interacting residues predicted by the Matrix of Local Coupling Energies method. Results show that the more efficient way to deal with the reranking problem is to consider the information given by the two protocols together, i.e. employing a consensus approach.

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.

Partition of the Reactive Species of the Suzuki-Miyaura Reaction between Aqueous and Micellar Environments.

The Suzuki-Miyaura reaction between the aryl halide (1) and the phenyl boronic acid (2), in the presence of the palladium(0) complex (3) as catalyst, gives the cross-coupling product (4) in quantitative yield when performed in basic aqueous solution of the nonionic surfactant Kolliphor-EL (K-EL). The partition between the aqueous and micellar environments of the species of this reaction has been investigated by means of Molecular Dynamics (MD) simulations. Starting from the K-EL molecules dispersed in water, a micelle model has been generated by MD simulations, adopting the 2016H66 force field. Reagent and product species have been described with the same force field, once the reliability of this force field has been tested comparing the n-octanol/water partition free energies calculated from the MD and Free Energy Perturbation (FEP) method with those obtained from the quantum-mechanical SMD method. The potential of mean force for the transfer process between water and the micellar phase of the different species has been calculated by the MD simulations and the Umbrella Sampling (US) method. The overall picture that emerges from these results confirms that the molecular species involved in this reaction prefers the micellar environment and concentrates in different but close zones of the micelle. This supports the experimental evidence that the use of suitable surfactant agents promotes reactivity, allowing micelles to behave as nanoreactors in which reactive species are solubilized and enhance their local concentration.

Can Water Act as a Nucleophile in CO Oxidation Catalysed by Mo/Cu CO-Dehydrogenase? Answers from Theory.

The aerobic CO dehydrogenase from Oligotropha carboxidovorans is an environmentally crucial bacterial enzyme for maintenance of subtoxic concentration of CO in the lower atmosphere, as it allows for the oxidation of CO to CO2 which takes place at its Mo-Cu heterobimetallic active site. Despite extensive experimental and theoretical efforts, significant uncertainties still concern the reaction mechanism for the CO oxidation. In this work, we used the hybrid quantum mechanical/molecular mechanical approach to evaluate whether a water molecule present in the active site might act as a nucleophile upon formation of the new C-O bond, a hypothesis recently suggested in the literature. Our study shows that activation of H2 O can be favoured by the presence of the Mo=Oeq group. However, overall our results suggest that mechanisms other than the nucleophilic attack by Mo=Oeq to the activated carbon of the CO substrate are not likely to constitute reactive channels for the oxidation of CO by the enzyme.

Dynamical Behavior and Conformational Selection Mechanism of the Intrinsically Disordered Sic1 Kinase-Inhibitor Domain.

Intrinsically Disordered Peptides and Proteins (IDPs) in solution can span a broad range of conformations that often are hard to characterize by both experimental and computational methods. However, obtaining a significant representation of the conformational space is important to understand mechanisms underlying protein functions such as partner recognition. In this work, we investigated the behavior of the Sic1 Kinase-Inhibitor Domain (KID) in solution by Molecular Dynamics (MD) simulations. Our results point out that application of common descriptors of molecular shape such as Solvent Accessible Surface (SAS) area can lead to misleading outcomes. Instead, more appropriate molecular descriptors can be used to define 3D structures. In particular, we exploited Weighted Holistic Invariant Molecular (WHIM) descriptors to get a coarse-grained but accurate definition of the variegated Sic1 KID conformational ensemble. We found that Sic1 is able to form a variable amount of folded structures even in absence of partners. Among them, there were some conformations very close to the structure that Sic1 is supposed to assume in the binding with its physiological complexes. Therefore, our results support the hypothesis that this protein relies on the conformational selection mechanism to recognize the correct molecular partners.

Role of the carbon defects in the catalytic oxygen reduction by graphite nanoparticles: a spectromagnetic, electrochemical and computational integrated approach.

The chemical groups present at the surface of graphite have been thought for a long time to be mainly responsible for its catalytic activity in the oxygen reduction reaction. Recently, it was proposed that the surface defects of graphite also significantly contribute to promote this reaction. Although the behaviour of surface defects has been reported, only few comments have been dedicated to their involvement in the mechanism and the possible intermediate species in the oxygen reduction reaction. Herein, we aim to present a more detailed explanation of the catalytic activity of graphite particles based on the structure of their defects and their size. Structural, spectroscopic and magnetic investigation (X-ray diffraction, Raman and electron spin resonance) and electrochemical measurements were performed to describe the nature of the defects and their aptitude to transfer electrons. Computational description supplied precise details of the energy of the different defects and their ability to promote the reduction, also suggesting the structure of the intermediate adduct in the oxygen reduction. The results indicated that molecular oxygen preferentially interacts with graphite defects, which involve the π-electron system and accumulation of the spin density on the edges of the grains, in particular, on the zig-zag edges present on ball-milled graphite. This promotes the reactivity of this nanomaterial. Furthermore, the activation increases by decreasing the particle size.

Quantum Stochastic Trajectories: The Fokker-Planck-Bohm Equation Driven by the Reduced Density Matrix.

The quantum molecular trajectory is the deterministic trajectory, arising from the Bohm theory, that describes the instantaneous positions of the nuclei of molecules by assuring the agreement with the predictions of quantum mechanics. Therefore, it provides the suitable framework for representing the geometry and the motions of molecules without neglecting their quantum nature. However, the quantum molecular trajectory is extremely demanding from the computational point of view, and this strongly limits its applications. To overcome such a drawback, we derive a stochastic representation of the quantum molecular trajectory, through projection operator techniques, for the degrees of freedom of an open quantum system. The resulting Fokker-Planck operator is parametrically dependent upon the reduced density matrix of the open system. Because of the pilot role played by the reduced density matrix, this stochastic approach is able to represent accurately the main features of the open system motions both at equilibrium and out of equilibrium with the environment. To verify this procedure, the predictions of the stochastic and deterministic representation are compared for a model system of six interacting harmonic oscillators, where one oscillator is taken as the open quantum system of interest. The undeniable advantage of the stochastic approach is that of providing a simplified and self-contained representation of the dynamics of the open system coordinates. Furthermore, it can be employed to study the out of equilibrium dynamics and the relaxation of quantum molecular motions during photoinduced processes, like photoinduced conformational changes and proton transfers.

The Challenging in silico Description of Carbon Monoxide Oxidation as Catalyzed by Molybdenum-Copper CO Dehydrogenase.

Carbon monoxide (CO) is a highly toxic gas to many living organisms. However, some microorganisms are able to use this molecule as the sole source of carbon and energy. Soil bacteria such as the aerobic Oligotropha carboxidovorans are responsible for the annual removal of about 2x108 tons of CO from the atmosphere. Detoxification through oxidation of CO to CO2 is enabled by the MoCu-dependent CO-dehydrogenase enzyme (MoCu-CODH) which-differently from other enzyme classes with similar function-retains its catalytic activity in the presence of atmospheric O2. In the last few years, targeted advancements have been described in the field of bioengineering and biomimetics, which is functional for future technological exploitation of the catalytic properties of MoCu-CODH and for the reproduction of its reactivity in synthetic complexes. Notably, a growing interest for the quantum chemical investigation of this enzyme has recently also emerged. This mini-review compiles the current knowledge of the MoCu-CODH catalytic cycle, with a specific focus on the outcomes of theoretical studies on this enzyme class. Rather controversial aspects from different theoretical studies will be highlighted, thus illustrating the challenges posed by this system as far as the application of density functional theory and hybrid quantum-classical methods are concerned.

Quantum stochastic trajectories: the Smoluchowski-Bohm equation.

Molecular systems are quantum systems, but the complete characterization of molecular motions within a fully quantum framework might appear to be an unfeasible task because it would require that the actual nuclear positions are established at any time. One would like to use a quantum molecular trajectory that defines the instantaneous nuclear positions and satisfies the predictions of quantum mechanics in terms of its statistical properties. Even though it can be proven that the single Bohm trajectory provides a representation of the quantum molecular trajectory, this solves the issue only on a theoretical ground: exact solutions of the Schrödinger-Bohm dynamical system are extremely computationally demanding. Therefore, we derive a stochastic equation of Smoluchowski type from the Schrödinger-Bohm dynamics, through projection operator techniques, in order to characterize the molecular motions of open quantum systems. The main quantum features of the motions emerge from the equilibrium distribution, i.e., the wave function's squared modulus integrated on the environment degrees of freedom. Furthermore, we verify the accuracy of the stochastic equation by comparing its predictions with those of the deterministic dynamics for a model system of six interacting harmonic oscillators. The indisputable advantage of this full quantum mechanical approach is that of representing the molecular dynamics, which controls important phenomena like vibrational relaxation, conformational transitions and activated processes, in a self consistent way and at the low computational cost of solving simple stochastic equations.

Thermal Pure States for Finite and Isolated Quantum Systems.

We study an ensemble of quantum pure states, the thermalization resilient ensemble (TRE), providing the statistical characterization of the thermal equilibrium of isolated quantum systems. Following a previous work where the ensemble was defined based on the invariance of the average populations upon thermal contact of identical systems, here we introduce a general methodology to generate quantum states according to the TRE statistic. The sampling is employed to characterize the ensemble distribution of thermodynamic functions like the entropy, internal energy, and temperature. The possibility of defining the temperature also for isolated quantum systems with a limited number of degrees of freedom is a distinctive feature of the TRE statistic which has no counterpart in other quantum statistical ensembles. The results are illustrated by explicit calculations for spin model systems.

Quantum Molecular Trajectory and Its Statistical Properties.

Despite the quantum nature of molecules, classical mechanics is often employed to describe molecular motions that play a fundamental role in a wide range of phenomena including chemical reactions. This is due to the need of assigning well-defined positions to the atomic nuclei during the time evolution of the system in order to describe unambiguously the molecular motions, whereas quantum mechanics provides information on probabilistic nature only. One would like to employ a quantum molecular trajectory that defines rigorously the instantaneous nuclear positions and, simultaneously, guarantees the conservation of all quantum mechanics predictions unlike the classical trajectory. We argue that such a quantum molecular trajectory can be formally defined and we prove that it corresponds to a single Bohm trajectory. Our analysis establishes a clear correspondence between the statistical properties of the trajectory and the quantum expectation values. The obvious and undeniable benefit is that of dealing with a quantum methodology fully characterizing the molecular motions without any reference to classical mechanics.

A theoretical study on the reactivity of the Mo/Cu-containing carbon monoxide dehydrogenase with dihydrogen.

The Mo/Cu-dependent CO dehydrogenase from Oligotropha carboxidovorans is an enzyme that is able to catalyze CO oxidation to CO2; moreover, it can also oxidize H2, thus eliciting a characteristic EPR signal. Interestingly, the Ag-substituted enzyme form proved unable to catalyze H2 oxidation. In the present contribution, we characterized the reactivity of the enzyme with H2 by quantum-chemical calculations. It was found that dihydrogen binding to the wild-type enzyme requires significant structural rearrangements of the active site Theoretical EPR spectra for plausible H2-bound models of the partially reduced, paramagnetic active site are also presented and compared with the experimental counterpart. Finally, density functional theory modeling shows that Ag substitution impairs H2 binding at the active site.

Quantum Statistical Ensemble Resilient to Thermalization.

The sampling of the wave function within a suitable ensemble is an important tool in the statistical analysis of a molecule interacting with its environment. The uniform statistical distribution of quantum pure states in an active space is often the privileged choice. However, such a distribution with constant average populations of eigenstates is not preserved upon the interaction between quantum systems. This appears as a severe methodological shortcoming, as long as a quantum system can be always considered as the result of interactions among previously isolated subsystems. In the present work we formulate an alternative statistical ensemble of pure states that is robust with respect to interaction, and it is thus preserved when subsystems are merged. It is derived from the condition of invariance of the average populations upon interaction between quantum systems in the same thermal state. These average populations allow a simple identification of the thermodynamic properties of the system. We find that such a statistical distribution is robust with respect to interaction of systems at different temperatures reproducing the thermalization of macroscopic bodies, and for this reason we identify it as the Thermalization Resilient Ensemble.

Influence of key amino acid mutation on the active site structure and on folding in acetyl-CoA synthase: a theoretical perspective.

Ad hoc quantum chemical modeling of the acetyl-CoA synthase local structure and folding allowed us to identify an unprecedented coordination mode of histidine sidechain to protein-embedded metal ions.

Quantum mechanical methods for the investigation of metalloproteins and related bioinorganic compounds.

It is well known that transition metal ions are often bound to proteins, conveying very specific functional properties. In fact, metalloproteins play crucial biological roles in the transport and activation of small molecules such as H2, O2, and N2, as well as in several other biochemical processes. However, even if the presence of transition metals in the active site of proteins allows a very rich biochemistry, the experimental disclosure of structure-activity relationships in metalloproteins is generally difficult exactly because of the presence of transition metals, which are intrinsically characterized by a very versatile and often elusive chemistry. For this reason, computational methods are becoming very popular tools in the characterization of metalloproteins. In particular, since computing power is becoming less and less expensive, due to the continuous technological development of CPUs, the computational tools suited to investigate metalloproteins are becoming more accessible and therefore more commonly used also in molecular biology and biochemistry laboratories. Here, we present the main procedures and computational methods based on quantum mechanics, which are commonly used to study the structural, electronic, and reactivity properties of metalloproteins and related bioinspired compounds, with a specific focus on the practical and technical aspects that must be generally tackled to properly study such biomolecular systems.

Computational Investigation on the Spectroscopic Properties of Thiophene Based Europium ß-Diketonate Complexes.

The adiabatic transition energies from the lowest triplet states of four Europium tris ß-diketonate/phenantroline complexes have been determined in vacuo and in dicholomethane solution by the ΔSCF approach at the density functional theory level, using the PBE1PBE and the CAM-B3LYP hybrid functionals. The calculated adiabatic transition energies have been compared with the experimental 0-0 transitions of each complex determined from phosphorescence spectra of the corresponding Gd(3+) complexes and followed by direct comparison between simulated and experimental spectra line shapes. For compound 1, the Eu(TTA)3Phen system, triplet states other than the lowest one and conformational isomers other than the one present in the crystallographic structure have been considered. In the crystallographic structure, this compound presents three quasi-degenerate low energy triplet states, differing for the TTA ligand where the two unpaired electrons are localized and showing close adiabatic transition energies. For compound 1, the lowest triplet states of the four investigated conformational isomers show similar characteristics and close adiabatic transition energies. On the basis of these results, an investigation of compounds 2-4 (Eu(Br-TTA)3Phen, Eu(DTDK)3Phen, and Eu(MeT-TTA)3) has been performed by considering only the isomer present in the crystallographic structure and only the lowest triplet state of each compound. For compounds 1-3, the energies of the lowest triplet states calculated by both functionals in solution including zero-point energy corrections well reproduce the experimental trends as well as the values of the adiabatic transition energies: CAM-B3LYP, the best performing functional, provides energies of the lowest triplet state with deviations from experiments lower than 1200 cm(-1). Also, the calculated vibrationally resolved phosphorescence spectra and UV-vis absorptions well reproduce the main features of their experimental counterparts. Significant differences between calculated and experimental results are observed for compound 4, for which difficulties in the experimental determination of the triplet state energy were encountered: our results show that the negligible photoluminescence quantum yield of this compound is due to the fact that the energy of the most stable triplet state is significantly lower than that of the resonance level of the Europium ion, and thus the energy transfer process is prevented. These results confirm the reliability of the adopted computational approach in calculating the energy of the lowest triplet state energy of these systems, a key parameter in the design of new ligands for lanthanide complexes presenting large photoluminescence quantum yields.

Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory.

[FeFe] hydrogenases are H2-evolving enzymes that feature a diiron cluster in their active site (the [2Fe]H cluster). One of the iron atoms has a vacant coordination site that directly interacts with H2, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O2. The [2Fe]H cluster can span various redox states. The active-ready form (Hox) attains the Fe(II)Fe(I) state. States more oxidized than Hox were shown to be inactive and/or resistant to O2. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O2, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe-N(azadithiolate) bond formation is favored for an Fe(II)Fe(II) active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding Fe(II)Fe(II) synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe]H cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H ox (inact) state.

Synthesis and characterization of a paramagnetic sialic acid conjugate as probe for magnetic resonance applications.

Magnetic Resonance Imaging (MRI) using paramagnetic systems as contrast agents is receiving increased attention as diagnostic tool in the clinic. At the same time, NMR of paramagnetic systems can also be applied in biochemical fields; for example, the use of Paramagnetic Relaxation Enhancement (PRE) allows structure refinement and the analysis of transient dynamic processes involved in macromolecular complex formation. Herein we report the synthesis and computational characterization of a new DOTA-like sialic acid conjugate, which can be used both in MRI and PRE applications when coordinated to a suitable paramagnetic metal.