Ari Zeida

Molecular basis of the mechanism of thiol oxidation by hydrogen peroxide in aqueous solution: challenging the SN2 paradigm

The oxidation of cellular thiol-containing compounds, such as glutathione and protein Cys residues, is considered to play an important role in many biological processes. Among possible oxidants, hydrogen peroxide (H(2)O(2)) is known to be produced in many cell types as a response to a variety of extracellular stimuli and could work as an intracellular messenger. This reaction has been reported to proceed through a S(N)2 mechanism, but despite its importance, the reaction is not completely understood at the atomic level. In this work, we elucidate the reaction mechanism of thiol oxidation by H(2)O(2) for a model methanethiolate system using state of the art hybrid quantum-classical (QM-MM) molecular dynamics simulations. Our results show that the solvent plays a key role in positioning the reactants, that there is a significant charge redistribution in the first stages of the reaction, and that there is a hydrogen transfer process between H(2)O(2) oxygen atoms that occurs after reaching the transition state. These observations challenge the S(N)2 mechanism hypothesis for this reaction. Specifically, our results indicate that the reaction is driven by a tendency of the slightly charged peroxidatic oxygen to become even more negative in the product via an electrophilic attack on the negative sulfur atom. This is inconsistent with the S(N)2 mechanism, which predicts a protonated sulfenic acid and hydroxyl anion as stable intermediates. These intermediates are not found. Instead, the reaction proceeds directly to unprotonated sulfenic acid and water.

Insights into the mechanism of the reaction between hydrogen sulfide and peroxynitrite.

Hydrogen sulfide and peroxynitrite are endogenously generated molecules that participate in biologically relevant pathways. A revision of the kinetic features of the reaction between peroxynitrite and hydrogen sulfide revealed a complex process. The rate constant of peroxynitrite decay, (6.65 ± 0.08) × 10(3) M(-1) s(-1) in 0.05 M sodium phosphate buffer (pH 7.4, 37°C), was affected by the concentration of buffer. Theoretical modeling suggested that, as in the case of thiols, the reaction is initiated by the nucleophilic attack of HS(-) on the peroxide group of ONOOH by a typical bimolecular nucleophilic substitution, yielding HSOH and NO2(-). In contrast to thiols, the reaction then proceeds to the formation of distinct products that absorb near 408 nm. Experiments in the presence of scavengers and carbon dioxide showed that free radicals are unlikely to be involved in the formation of these products. The results are consistent with product formation involving the reactive intermediate HSSH and its fast reaction with a second peroxynitrite molecule. Mass spectrometry and UV-Vis absorption spectra predictions suggest that at least one of the products is HSNO2 or its isomer HSONO.

Mechanism of cysteine oxidation by peroxynitrite: an integrated experimental and theoretical study

Since peroxynitrite was identified as a pathophysiological agent it has been implicated in a great variety of cellular processes. Particularly, peroxynitrite mediated oxidation of cellular thiol-containing compounds such as Cys residues, is a key event which has been extensively studied. Although great advances have been accomplished, the reaction is not completely understood at the atomic level. Aiming to shed light on this subject, we present an integrated kinetic and theoretical study of the oxidation of free Cys by peroxynitrite. We determined pH-independent thermodynamic activation parameters, namely those corresponding to the reaction between the reactive species: Cys thiolate and peroxynitrous acid. We found a pH-independent activation energy of 8.2 ± 0.6 kcal/mol. Simulations were performed using state of the art hybrid quantum-classical (QM-MM) molecular dynamics simulations. Our results are consistent with a SN2 mechanism, with Cys sulfenic acid and nitrite anion as products. The activation barrier is mostly due to the alignment of sulfurs thiolate atom with the oxygen atoms of the peroxide, along with the concomitant charge reorganization and important changes in the solvation profile. This work provides an atomic detailed description of the reaction mechanism and a framework to understand the environment effects on peroxynitrite reactivity with protein thiols.

Nitrosodisulfide [S2NO](-) (perthionitrite) is a true intermediate during the "cross-talk" of nitrosyl and sulfide.

Nitrosodisulfide S2NO- is a controversial intermediate in the reactions of S-nitrosothiols with HS- that produce NO and HNO. QM-MM molecular dynamics simulations combined with TD-DFT analysis contribute to a clear identification of S2NO- in water, acetone and acetonitrile, accounting for the UV-Vis signatures and broadening the mechanistic picture of N/S signaling in biochemistry.

Molecular Basis of Hydroperoxide Specificity in Peroxiredoxins: The Case of AhpE from Mycobacterium tuberculosis

Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts ∼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzymes dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.

PrxQ B from Mycobacterium tuberculosis is a monomeric, thioredoxin-dependent and highly efficient fatty acid hydroperoxide reductase

Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M-1s-1, respectively), while the rate constant of peroxynitrite reduction was (0.6-1.4) ×106M-1s-1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106 and 1×106M-1s-1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)

Significance Trypanosoma cruzi, the causative agent of Chagas disease, affects 8–10 million people in Latin America. Parasite antioxidant systems are essential for parasite survival and infectivity in the vertebrate host. Herein, we characterized the enzymic properties, subcellular localization, and contribution to parasite virulence of a T. cruzi hybrid type A member of class I heme peroxidases. The enzyme reacts fast with hydrogen peroxide and utilizes both ferrocytochrome c and ascorbate as reducing substrates [T. cruzi ascorbate peroxidase (TcAPx)-cytochrome c peroxidase (CcP)]. A unique subcellular distribution of TcAPx-CcP in the infective stages suggests a role during parasite–host interactions. Infection of macrophages and cardiomyocytes, as well as in mice, confirmed the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium. The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M−1⋅s−1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M−1⋅s−1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host–parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

Chemical Reactivity and Spectroscopy Explored From QM/MM Molecular Dynamics Simulations Using the LIO Code

In this work we present the current advances in the development and the applications of LIO, a lab-made code designed for density functional theory calculations in graphical processing units (GPU), that can be coupled with different classical molecular dynamics engines. This code has been thoroughly optimized to perform efficient molecular dynamics simulations at the QM/MM DFT level, allowing for an exhaustive sampling of the configurational space. Selected examples are presented for the description of chemical reactivity in terms of free energy profiles, and also for the computation of optical properties, such as vibrational and electronic spectra in solvent and protein environments.

Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations

Glutamine synthetase is an important enzyme that catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. In mammals, it plays a key role in preventing excitotoxicity in the brain and detoxifying ammonia in the liver. In plants and bacteria, it is fundamental for nitrogen metabolism, being critical for the survival of the organism. In this work, we show how the use of classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics simulations allowed us to examine the structural properties and dynamics of human glutamine synthetase (HsGS), as well as the reaction mechanisms involved in the catalytic process with atomic level detail. Our results suggest that glutamine formation proceeds through a two-step mechanism that includes a first step in which the γ-glutamyl phosphate intermediate forms, with a 5 kcal/mol free energy barrier and a -8 kcal/mol reaction free energy, and then a second rate-limiting step involving the ammonia nucleophilic attack, with a free energy barrier of 19 kcal/mol and a reaction free energy of almost zero. A detailed analysis of structural features within each step exposed the relevance of the acid-base equilibrium related to protein residues and substrates in the thermodynamics and kinetics of the reactions. These results provide a comprehensive study of HsGS dynamics and establish the groundwork for further analysis regarding changes in HsGS activity, as occur in natural variants and post-translational modifications.