Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme

Modélisation du mécanisme catalytique de l'urate oxydase

In this work, a theoretical study has been carried our in order to understand the catalytic mechanism of Uox. i) study of the physical and chemical properties of the uric acid and its anions; ii) determination of the intrinsic reactivity of the uric acid and its anions with dioxygen; iii) determination of the urate form bound by the enzyme and its reactivity in an active site model. All these studies were carried out at various levels of quantum mechanics, including semi-empirical, DFT and ab initio (HF and MP2) methods. Our study highlights two significant peculiarities of the catalytic mechanism of urate oxidase; i) the substrate bound to the enzyme is the 3-7 urate dianion form, which is not the most stable dianion form in solution but the second most one; ii) the reaction pathway displays a continuous spin change from the reactive state, a triplet state, to the final state, a single state. This spin change occurs without any photon emission but through a degenerescency between the singlet and the triplet states along the first steps of the reaction.L'objectif de ce travail est de comprendre le mécanisme catalytique de l'urate oxydase à l'aide des méthodes de la chimie quantique. Pour cela nous avons étudié ce mécanisme en trois étapes : i) étude des propriétés physico-chimiques de l'acide urique et de ses anions ; ii) étude de la réactivité intrinsèque de l'acide urique et de ses anions avec le dioxygène ; iii) étude de la réactivité du substrat réel dans un modèle de site actif. L'ensemble de ces études a été effectué selon différents niveaux de mécanique quantique : semi-empirique, DFT ou encore ab initio (niveaux HF et MP2). L'ensemble de nos études a permis de mettre en évidence au moins deux particularités très importantes du mécanisme catalytique de l'urate oxydase : i) le substrat reconnu par l'enzyme est dianionique et ne correspond pas au dianion le plus stable en solution mais à la deuxième espèce la plus stable ; ii) le chemin réactionnel implique un changement d'état de spin de l'état réactif (triplet) à l'état final (singulet). Ce changement d'état de spin intervient sans transition d'état (i.e. sans émission de proton)

Modélisation du mécanisme catalytique de l'urate oxydase

L'objectif de ce travail est de comprendre le mécanisme catalytique de l'urate oxydase à l'aide des méthodes de la chimie quantique. Pour cela nous avons étudié ce mécanisme en trois étapes : i) étude des propriétés physico-chimiques de l'acide urique et de ses anions ; ii) étude de la réactivité intrinsèque de l'acide urique et de ses anions avec le dioxygène ; iii) étude de la réactivité du substrat réel dans un modèle de site actif. L'ensemble de ces études a été effectué selon différents niveaux de mécanique quantique : semi-empirique, DFT ou encore ab initio (niveaux HF et MP2). L'ensemble de nos études a permis de mettre en évidence au moins deux particularités très importantes du mécanisme catalytique de l'urate oxydase : i) le substrat reconnu par l'enzyme est dianionique et ne correspond pas au dianion le plus stable en solution mais à la deuxième espèce la plus stable ; ii) le chemin réactionnel implique un changement d'état de spin de l'état réactif (triplet) à l'état final (singulet). Ce changement d'état de spin intervient sans transition d'état (i.e. sans émission de proton).In this work, a theoretical study has been carried our in order to understand the catalytic mechanism of Uox. i) study of the physical and chemical properties of the uric acid and its anions; ii) determination of the intrinsic reactivity of the uric acid and its anions with dioxygen; iii) determination of the urate form bound by the enzyme and its reactivity in an active site model. All these studies were carried out at various levels of quantum mechanics, including semi-empirical, DFT and ab initio (HF and MP2) methods. Our study highlights two significant peculiarities of the catalytic mechanism of urate oxidase; i) the substrate bound to the enzyme is the 3-7 urate dianion form, which is not the most stable dianion form in solution but the second most one; ii) the reaction pathway displays a continuous spin change from the reactive state, a triplet state, to the final state, a single state. This spin change occurs without any photon emission but through a degenerescency between the singlet and the triplet states along the first steps of the reaction.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

A theoretical investigation of the CO2-philicity of amides and carbamides

International audienc

Taste for Chiral Guests: Investigating the Stereoselective Binding of Peptides to β-Cyclodextrins

International audienc

Cavity Closure Dynamics of Peracetylated β-Cyclodextrins in Supercritical Carbon Dioxide

Structural properties of peracetylated β-cyclodextrin in supercritical carbon dioxide were investigated by means of molecular dynamics simulations. The study indicated a strong reduction of the cavity accessibility to guest molecules, compared to native β-cyclodextrin in water. Indeed, the cavity is self-closed during the largest part of the simulation, which agrees well with suggestions made on the basis on high-pressure NMR experiments. Self-closure happens because one glucose unit undergoes a main conformational change (from chair to skew) that brings one of the acetyl groups in the wide rim of the cyclodextrin to the cavity interior. This arrangement turns out to be quite favorable, persisting for several nanoseconds. In addition to the wide rim self-closure, a narrow rim self-closure may also occur, though it is less likely and exhibits short duration (<1 ns). Therefore, the number of solvent molecules reaching the cavity interior is much smaller than that found in the case of native β-cyclodextrin in water after correction to account for different molar densities. These findings support the weak tendency of the macromolecule to form host–guest complexes in this nonconventional medium, as reported by some experiments. Finally, Lewis acid/base interactions between the acetyl carbonyl groups and the solvent CO₂ molecules were analyzed through ab initio calculations that revealed the existence of a quite favorable four-member ring structure not yet reported. The ensemble of these results can contribute to establish general thermodynamic principles controlling the formation of inclusion complexes in supercritical CO₂, where the hydrophilicity/hydrophobicity balance is not applicable

Driving Forces Controlling Host-Guest Recognition in Supercritical Carbon Dioxide Solvent.

International audienceThe formation of supramolecular host-guest complexes is a very useful and widely employed tool in chemistry. However, supramolecular chemistry in non-conventional solvents such as supercritical carbon dioxide (scCO2 ), one of the most promising sustainable solvents, is still in its infancy. In this work, we explored a successful route to the development of green processes in supercritical CO2 by combining a theoretical approach with experiments. We were able to synthesize and characterize an inclusion complex between a polar aromatic molecule (benzoic acid) and peracetylated-β-cyclodextrin, which is soluble in the supercritical medium. This finding opens the way to wide, environmental friendly, applications of scCO2 in many areas of chemistry, including supramolecular synthesis, reactivity and catalysis, micro and nano-particle formation, molecular recognition, as well as enhanced extraction processes with increased selectivity

Taste for Chiral Guests: Investigating the Stereoselective Binding of Peptides to β‑Cyclodextrins

Obtaining compounds of diastereomeric purity is extremely important in the field of biological and pharmaceutical industry, where amino acids and peptides are widely employed. In this work, we theoretically investigate the possibility of chiral separation of peptides by β-cyclodextrins (β-CDs), providing a description of the associated interaction mechanisms by means of molecular dynamics (MD) simulations. The formation of host/guest complexes by including a model peptide in the macrocycle cavity is analyzed and discussed. We consider the terminally blocked phenylalanine dipeptide (Ace-Phe-Nme), in the l- and d-configurations, to be involved in the host/guest recognition process. The CD–peptide free energies of binding for the two enantiomers are evaluated through a combined approach that assumes: (1) extracting a set of independent molecular structures from the MD simulation, (2) evaluating the interaction energies for the host/guest complexes by hybrid quantum mechanics/molecular mechanics (QM/MM) calculations carried out on each structure, for which we also compute, (3) the solvation energies through the Poisson–Boltzmann surface area method. We find that chiral discrimination by the CD macrocycle is of the order of 1 kcal/mol, which is comparable to experimental data for similar systems. According to our results, the Ace-(d)­Phe-Nme isomer leads to a more stable complex with a β-CD compared to the Ace-(l)­Phe-Nme isomer. Nevertheless, we show that the chiral selectivity of β-CDs may strongly depend on the secondary structure of larger peptides. Although the free energy differences are relatively small, the predicted selectivities can be rationalized in terms of host/guest hydrogen bonds and hydration effects. Indeed, the two enantiomers display different interaction modes with the cyclodextrin macrocavity and different mobility within the cavity. This finding suggests a new interpretation for the interactions that play a key role in chiral recognition, which may be exploited to design more efficient and selective chiral separations of peptides

X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase

International audienceUrate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid using gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide in absence of any cofactor or transition metal. The catalytic mechanism was investigated using X-ray diffraction, electron spin resonance spectroscopy (ESR), and quantum mechanics calculations. The X-ray structure of the anaerobic enzyme-substrate complex gives credit to substrate activation before the dioxygen fixation in the peroxo hole, where incoming and outgoing reagents (dioxygen, water, and hydrogen peroxide molecules) are handled. ESR spectroscopy establishes the initial monoelectron activation of the substrate without the participation of dioxygen. In addition, both X-ray structure and quantum mechanic calculations promote a conserved base oxidative system as the main structural features in UOX that protonates/deprotonates and activate the substrate into the doublet state now able to satisfy the Wigner's spin selection rule for reaction with molecular oxygen in its triplet ground state