Molybdenum Trihydride Complexes: Computational Model of Oxidatively Induced Reductive Elimination of Dihydrogen.

Recent experimental work shows that the 18-electron molybdenum complexes (1,2,4-C5H2tBu3)Mo(PMe3)2H3 (CptBuMoH3) and (C5HiPr4)Mo(PMe3)2H3 (CpiPrMoH3) undergo oxidatively induced reductive elimination of dihydrogen (H2), slowly forming the 15-electron monohydride species in tetrahydrofuran and acetonitrile. The 17-electron [CptBuMoH3]+ derivative was stable enough to be characterized by X-ray diffraction, while [CpiPrMoH3]+ was not. Density functional theory calculations of the H2 elimination pathways for both complexes in the gas phase and in a continuum solvent model indicate that H2 elimination from [CpiPrMoH3]+ has a lower barrier than that from [CptBuMoH3]+. Further, a specific solvent association, which is stronger for [CptBuMoH3]+ than for [CpiPrMoH3]+, contributes to the stability of the former. In agreement with the experimental observations, the calculations predict that [CptBuMoH3]+ would be in a quartet state at room temperature and a doublet state at 4.2 K, while [CpiPrMoH3]+ is in a doublet state even at room temperature.

Molybdenum Trihydride Complexes: Computational Determinations of Hydrogen Positions and Rearrangement Mechanisms.

In crystal structures of the molybdenum complexes [(1,2,4-C5H2(t)Bu3)Mo(PMe3)2H3] (Cp(t)Bu3) and [(C5H(i)Pr4)Mo(PMe3)2H3] (Cp(i)Pr4), the Mo-bound hydrogen positions were resolved for Cp(t)Bu3, but not for Cp(i)Pr4. NMR experiments revealed the existence of an unknown mechanism for hydrogen atom exchange in these molecules, which can be "frozen out" for Cp(t)Bu3 but not for Cp(i)Pr4. Density functional theory calculations of the most stable conformations for both complexes in the gas phase and in a continuum solvent model indicate that the H's of the Cp(i)Pr4 complex are unresloved because of their disorder, which does not occur for Cp(t)Bu3. A corresponding examination of alternative rearrangement pathways shows that the rearrangements of the H's could occur by two mechanisms: parallel to the cyclopentadienyl (Cp) ring in a single step and perpendicular to the Cp ring in two steps. The parallel pathway is preferred for both molecules, but it has a lower energy barrier for Cp(i)Pr4 than for Cp(t)Bu3.

Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: Horseradish peroxidase and dehaloperoxidase.

The mechanism of the dehalogenation step catalyzed by dehaloperoxidase (DHP) from Amphitrite ornata, an unusual heme-containing protein with a globin fold and peroxidase activity, has remarkable similarity with that of the classical heme peroxidase, horseradish peroxidase (HRP). Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, we have concluded that two sequential one electron oxidations of the halogenated phenol substrate leads to a cationic intermediate that strongly resembles a Meisenheimer intermediate - a commonly formed reactive complex during nucleophilic aromatic substitution reactions especially in the case of arenes carrying electron withdrawing groups.

Modeling the Mechanical Response of Microtubule Lattices to Pressure.

Microtubules, the largest and stiffest filaments of the cytoskeleton, have to be well adapted to the high levels of crowdedness in cells to perform their multitude of functions. Furthermore, fundamental processes that involve microtubules, such as the maintenance of the cellular shape and cellular motion, are known to be highly dependent on external pressure. In light of the importance of pressure for the functioning of microtubules, numerous studies interrogated the response of these cytoskeletal filaments to osmotic pressure, resulting from crowding by osmolytes, such as poly(ethylene glycol)/poly(ethylene oxide) (PEG/PEO) molecules, or to direct applied pressure. The interpretation of experiments is usually based on the assumptions that PEG molecules have unfavorable interactions with the microtubule lattices and that the behavior of microtubules under pressure can be described by using continuous models. We probed directly these two assumptions. First, we characterized the interaction between the main interfaces in a microtubule filament and PEG molecules of various sizes using a combination of docking and molecular dynamics simulations. Second, we studied the response of a microtubule filament to compression using a coarse-grained model that allows for the breaking of lattice interfaces. Our results show that medium length PEG molecules do not alter the energetics of the lateral interfaces in microtubules but rather target and can penetrate into the voids between tubulin monomers at these interfaces, which can lead to a rapid loss of lateral interfaces under pressure. Compression of a microtubule under conditions corresponding to high osmotic pressure results in the formation of the deformed phase found in experiments. Our simulations show that the breaking of lateral interfaces, rather than the buckling of the filament inferred from the continuous models, accounts for the deformation.

Mechanics of the Microtubule Seam Interface Probed by Molecular Simulations and in Vitro Severing Experiments.

Microtubules (MTs) are structural components essential for cell morphology and organization. It has recently been shown that defects in the filament's lattice structure can be healed to create stronger filaments in a local area and ultimately cause global changes in MT organization and cell mobility. The ability to break, causing a defect, and heal appears to be a physiologically relevant and important feature of the MT structure. Defects can be created by MT severing enzymes and are target sites for complete severing or for healing by newly incorporated dimers. One particular lattice defect, the MT lattice ''seam" interface, is a location often speculated to be a weak site, a site of disassembly, or a target site for MT binding proteins. Despite seams existing in many MT structures, very little is known about the seam's role in MT function and dynamics. In this study, we probed the mechanical stability of the seam interface by applying coarse-grained indenting molecular dynamics. We found that the seam interface is as structurally robust as the typical lattice structure of MTs. Our results suggest that, unlike prior results that claim the seam is a weak site, it is just as strong as any other location on the MT, corroborating recent mechanical measurements.

Proof of Principle that Molecular Modeling Followed by a Biophysical Experiment Can Develop Small Molecules that Restore Function to the Cardiac Thin Filament in the Presence of Cardiomyopathic Mutations.

This article reports a coupled computational experimental approach to design small molecules aimed at targeting genetic cardiomyopathies. We begin with a fully atomistic model of the cardiac thin filament. To this we dock molecules using accepted computational drug binding methodologies. The candidates are screened for their ability to repair alterations in biophysical properties caused by mutation. Hypertrophic and dilated cardiomyopathies caused by mutation are initially biophysical in nature, and the approach we take is to correct the biophysical insult prior to irreversible cardiac damage. Candidate molecules are then tested experimentally for both binding and biophysical properties. This is a proof of concept study-eventually candidate molecules will be tested in transgenic animal models of genetic (sarcomeric) cardiomyopathies.

Measurement and Prediction of Chlorine Kinetic Isotope Effects in Enzymatic Systems.

Approaches to determine chlorine kinetic isotope effects (Cl-KIEs) on enzymatic dehalogenations are discussed and illustrated by representative examples. Three aspects are considered. First methodology for experimental measurement of Cl-KIEs, with stress being on FAB-IRMS technique developed in our laboratory, is described. Subsequently, we concentrate our discussion on the consequences of reaction complexity in the interpretation of experimental values, a problem especially important in cases of polychlorinated reactants. The most fruitful studies of enzymatic dehalogenations by Cl-KIEs require their theoretical evaluation, hence the computational focus of the second part of this chapter.

A computational study on enzymatically driven oxidative coupling of chlorophenols: an indirect dehalogenation reaction.

Density functional theory calculations have been used to describe the mechanism of the dimerization reaction catalyzed by peroxidases and predict whether it could be accompanied by chlorine isotopic fractionation when various chlorophenols are used as their substrates. Since free radicals formed during the catalytic cycle of peroxidases can undergo coupling reactions (either radical-radical or radical-molecule) that can lead to dimers and also polymers formation four different pathways have been considered: radical-anion, radical-cation, radical-radical (singlet state) and radical-radical (triplet state). The following substrates have been investigated: 2-chlorophenol, 4-chlorophenol, 2,4,6-trichlorophenol and 4-chloro-2,6-dimethylphenol. Based on the obtained energetic profiles radical-cation and radical-radical (singlet) coupling seem to be the most probable. Radical-anion coupling although energetically more expensive should not be disregarded taking into account the excess of the anionic form of the substrate being provided in the course of enzymatic reaction. Upon dimer formation halogen is released during radical-anion and radical-radical triplet coupling. However only the latter pathway exhibits chlorine isotopic fractionation. Radical-cation and radical-radical singlet couplings are two-step reactions where the second step comprises intramolecular chlorine transfer accompanied by large chlorine isotope effect.

Kinetic isotope effects on dehalogenations at an aromatic carbon.

In order to interpret the observed isotopic fractionation it is necessaryto understand its relationship with the isotope effect(s) on steps that occur during the conversion of the initial reactant to the final product. We examine this relationship from the biochemical point of view and elaborate on the consequences of the assumptions that it is based on. We illustrate the discrepancies between theoretical and experimental interpretation of kinetic isotope effects on examples of dehalogenation reactions that occur at an aromatic carbon atom. The examples include 4-chlorobenzoyl-CoA dehalogenase-catalyzed conversion of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA, dehaloperoxidase-catalyzed conversion of 2,4,6-trichlorophenol to 2,6-dichloroquinone, and spontaneous hydrolysis of atrazine at pH 12. For this latter reaction we have measured the chlorine kinetic isotope effect and estimated its value theoretically at the DFT level of theory. Results of chlorine kinetic isotope effects suggest that the studied dechlorination reactions proceed in a single step with significant weakening of the carbon-chlorine bond in the transition state.

Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects.

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.