Role of Microsolvation and Quantum Effects in the Accurate Prediction of Kinetic Isotope Effects: The Case of Hydrogen Atom Abstraction in Ethanol by Atomic Hydrogen in Aqueous Solution

Hydrogen abstraction from ethanol by atomic hydrogen in aqueous solution is studied using two theoretical approaches: the multipath variational transition state theory (MP-VTST) and a path-integral formalism in combination with free-energy perturbation and umbrella sampling (PI-FEP/UM). The performance of the models is compared to experimental values of H kinetic isotope effects (KIE). Solvation models used in this study ranged from purely implicit, via mixed–microsolvation treated quantum mechanically via the density functional theory (DFT) to fully explicit representation of the solvent, which was incorporated using a combined quantum mechanical-molecular mechanical (QM/MM) potential. The effects of the transition state conformation and the position of microsolvating water molecules interacting with the solute on the KIE are discussed. The KIEs are in good agreement with experiment when MP-VTST is used together with a model that includes microsolvation of the polar part of ethanol by five or six water molecules, emphasizing the importance of explicit solvation in KIE calculations. Both, MP-VTST and PI-FEP/UM enable detailed characterization of nuclear quantum effects accompanying the hydrogen atom transfer reaction in aqueous solutionThis work was partially supported by the National Science Center in Poland (Sonata BIS grant UMO-2014/14/E/ST4/00041) and in part by PLGrid Infrastructure (Poland). S.K. acknowledges the Erasmus+ programme within which his 3-month project conducted at the University of Santiago de Compostela was possible. A.F-.R. thanks the Consellería de Cultura, Educación e Ordenación Universitaria (Axuda para Consolidación e Estructuración de unidades de investigación competitivas do Sistema Universitario de Galicia, Xunta de Galicia ED431C 2017/17 & Centro singular de investigación de Galicia acreditación 2016-2019, ED431G/09) and the European Regional Development Fund (ERDF). D.F-.C. also thanks Xunta de Galicia for financial support through a postdoctoral grantS

Biochemical and Mechanistic Studies of Nitronate Monooxygenase and Roles of Histidine Residues in Select Flavoprotein Oxidases

Nitronate monooxygenase (NMO) catalyzes the flavin-dependent oxidation of propionate 3-nitronate (P3N) via the formation of an anionic flavosemiquinone. The oxidation of substrate includes the formation of a peroxy-nitro acid intermediate. P3N is activated to its radical form via a single electron transfer onto the FMN cofactor forming the anionic flavosemiquinone. Reoxidation of FMN cofactor from the anionic semiquinone has been proposed to go through two routes dependent upon which radical species oxygen reacts with first, radical P3N or the semiquinone. The recent crystallographic determination of NMO from Cyberlindnera saturnus and steady-state kinetics revealed an allosteric activation effect on the enzyme by PEG 3350 with respect to P3N. Choline oxidase (CHO) catalyzes the two-step oxidation of choline to glycine betaine via an enzyme-bound FAD cofactor. In the first redox reaction, choline is activated to it alkoxide form by means of an enzyme-derived catalytic base, H466. This histidine residue has been shown to not only act as a general base but an electrostatic catalyst stabilizing the negative charge accumulated on the reduced flavin species as shown by replacing the residue with alanine, aspartate, and glutamine using site-directed mutagenesis. CHO was also observed to catalyze excited state reactions as facilitated by H466. Evidence for the ESR comes from the observation of a pL-dependence on the fluorescence emission of CHO in H2O and D2O. Using fluorescence spectroscopy and pH effects, a hydroxy-C4a flavin intermediate was detected in the wild-type and S101A variant with and without oxygen indicating the adduct formation was with an active site hydroxide ion. The mechanism of formation has been elucidated

On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase

Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme. The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not. In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine. The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 1,1-[2H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase

Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter Globiformis

The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions

Systematic methods for solvent design : towards better reactive processes

The focus of this thesis is the development of novel methodologies for systematic identification of optimal solvents for chemical reactions. Two aspects are considered: the integrated solvent and process design using a mixed solvent, and the design of an optimal solvent using ab initio methods that do not rely on experimental data. A methodology is developed for the integrated design of a CO2-expanded solvent in a reaction process. Posing as objective function the cost of the process, for a defined production rate, an optimisation problem is formulated, with decision variables that include the organic co-solvent, the composition and the mass of the mixed solvent. Emphasis is placed on the prediction of the reaction rate, for which the solvatochromic equation combined with a preferential solvation model are used, and on solid-vapour-liquid phase equilibrium, for which the group-contribution volume translated Peng-Robinson equation of state is used. The proposed methodology is applied to the Diels-Alder reaction of anthracene and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and three CO2-expanded solvents are considered (acetone, acetonitrile and methanol). Acetonitrile and acetone are found to offer good performance over a range of CO2 concentrations. The importance of taking into account multiple process performance indicators, when designing gas-expanded liquids, is highlighted. As a further step toward systematic solvent design approaches that are not limited by the availability of experimental data and consider a large number of candidate solvents, an ab initio methodology is developed for the design of optimal solvents for reactions. The developed method combines quantum mechanical calculations with a computer-aided molecular design formulation. In order to limit the number of QM calculations but also retain accuracy and ensure convergence, the Kriging approach is used. Kriging is a response surface approach, which has recently attracted a lot of attention because it is an exact extrapolator with a statistical interpretation which makes it stand out from other methods. The proposed approach is used successfully to identify promising solvents for the Menschutkin reaction of phenacyl bromide and pyridine and the Cope elimination of methylamine oxide. The use of Kriging as the surrogate model is found to lead to improved solvents when compared to the simpler solvatochromic equation used in previous work.Open Acces

Investigating Novel Methods of Interaction with Pharmaceutically Relevant Enzymes

Metalloproteins requiring one or more metal ions for normal function make up 30% of all known proteins, and many critical biological pathways contain at least one metallo-enzyme. Di-nuclear metallo-proteins constitute a large class of these proteins yet we currently lack effective methods of inhibiting these enzymes for the development of new medical therapies, particularly for the discovery of new antibiotics. Our work has focused on developing novel functionalities that selectively interact with di-nuclear catalytic centers, and we are targeting three separate di-zinc-metallo-enzymes that are unique to bacteria and play key roles in their growth and development. These enzymes are DapE, AiiA, and NDM-1. DapE is involved in biosynthesis of lysine and meso-diaminopimelic acid, essential precursors in the production of bacterial cell walls. AiiA is a di-Zn-dependent lactonase involved in bacterial cell-cell communication, and NDM-1 is a di-metallo-beta-lactamase capable of deactivating the most commonly administered antibiotics, gaining international attention for this enzyme as a clinically-relevant pharmaceutical target, yet drug development efforts have proven ineffective due to a lack of effective inhibitors. As part of our ongoing studies to functionally annotate the Gcn5-related N-acetyltransferase (GNAT) PA4794 from Pseudomonas aeruginosa with unknown functions, we have used PA4794 as a model system for exploring efficient formation of bisubstrate complexes to enhance our success rate in obtaining co-crystal structures of GNATs with ligands bound in their acceptor sites. We have synthesized and tested substrate analogs of the previously identified N-phenylacetyl glycine lysine (NPAcGK) enabling two separate three-dimensional structures of PA4794 with NPAcGK analog-derived bisubstrates formed through direct reaction with CoA—the first through direct alkylation with a reactive substrate, and the second through X-ray induced radical-mediated process. We have also performed docking and molecular dynamics simulations of the reverse reaction pathway from the NPAcGK product back to formation of the tetrahedral intermediate/transition state to complement our structural work and to explore the key ligand-protein interactions within the active site of PA4794, guiding mutant synthesis and kinetics to explore the role of key residues in the active site

ANION PHOTOELECTRON SPECTROSCOPIC STUDIES: FROM BIOMOLECULES, TO SIMPLE ORGANICS, TO METAL COMPOUNDS

Photoelectron spectroscopic studies on gas phase mass-selected anions were performed on a variety of molecular systems. These studies are grouped by themes culminating into five chapters discussing biomolecules, electron induced proton transfer, alkoxides, metal oxides, metal cluster reactivity, and electron binding to aromatic molecules. These experiments were primarily performed in the Bowen lab at Johns Hopkins University using our continuous negative ion photoelectron spectrometer. The metal oxide studies were performed on our pulsed negative ion photoelectron spectrometer while the metal cluster reactivity studies were carried out on location at Karlsruhe institute of Technology (KIT), Germany under Professor Hansgeorg Schnӧckel using an FT-ICR mass spectrometer. The study of nucleic acid bases (nucleobases) and their interaction with low energy electrons elucidates a better molecular-level understanding of radiation induced mutagenesis (Chapter 1). Additionally, studies of hydrated and rare-gas solvated nucleobase anions provide strong evidence supporting the co-existence of dipole-bound and valence anion states. Modified uracil analogues 6-azauracil and 5-substituted derivatives are discussed in relation to their properties as radiosensitizers. Electron-induced proton transfer studies (Chapter 2) yields information on the most fundamental processes in chemistry. Intramolecular hydrogen bonding is important to the aforementioned biomolecule systems, but carrying out experiments on the systems acetoacetic acid and oxalic acid allow for better insight utilizing molecules with a larger vapor pressure. The system of 1,8-bis(dimethylamino)naphthalene, HCl and an excess electron inducing intermolecular proton transfer presents a novel acid-base interaction that demonstrates the fundamental processes of how salt complexes are formed. Chapter 3 presents the adiabatic electron affinities and vertical detachment energies of a series of deprotonated alcohols, also referred to as alkoxides. This information yields O-H bond dissociation energies of the corresponding alcohol using the gas phase acidities of the associated alcohol of interest and the thermochemical cycle. Zirconium and hafnium transition metal oxides presented in Chapter 4 examines metals with similar physical properties do not necessarily form oxides that will also exhibit similar properties. Reactivity studies were performed with Al/Ga13¯ and surrounding cluster sizes reacting with O2. This is to further study the odd/even reactivity effect and improve characterization of these clusters, Al/Ga13¯ specifically, for use in cluster-assembled materials. Finally, binding of an excess electron to several aromatic systems are discussed in Chapter 5. Benzaldehyde exhibits a unique spectral profile as a result of vibrational progressions from electron attachment. p-Nitroaniline and the chiral molecules of N-paranitrophenylsulfonylalanine and N-paranitrophenylalanine were studied to determine how the smaller molecular components may affect the physical properties of the larger molecular complex