Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.

Assessment of Embedding Schemes in a Hybrid Machine Learning/Classical Potentials (ML/MM) Approach.

Machine learning (ML) methods have reached high accuracy levels for the prediction of in vacuo molecular properties. However, the simulation of large systems solely through ML methods (such as those based on neural network potentials) is still a challenge. In this context, one of the most promising frameworks for integrating ML schemes in the simulation of complex molecular systems are the so-called ML/MM methods. These multiscale approaches combine ML methods with classical force fields (MM), in the same spirit as the successful hybrid quantum mechanics-molecular mechanics methods (QM/MM). The key issue for such ML/MM methods is an adequate description of the coupling between the region of the system described by ML and the region described at the MM level. In the context of QM/MM schemes, the main ingredient of the interaction is electrostatic, and the state of the art is the so-called electrostatic-embedding. In this study, we analyze the quality of simpler mechanical embedding-based approaches, specifically focusing on their application within a ML/MM framework utilizing atomic partial charges derived in vacuo. Taking as reference electrostatic embedding calculations performed at a QM(DFT)/MM level, we explore different atomic charges schemes, as well as a polarization correction computed using atomic polarizabilites. Our benchmark data set comprises a set of about 80k small organic structures from the ANI-1x and ANI-2x databases, solvated in water. The results suggest that the minimal basis iterative stockholder (MBIS) atomic charges yield the best agreement with the reference coupling energy. Remarkable enhancements are achieved by including a simple polarization correction.

Insights into the product release mechanism of dengue virus NS3 helicase.

The non-structural protein 3 helicase (NS3h) is a multifunctional protein that is critical in RNA replication and other stages in the flavivirus life cycle. NS3h uses energy from ATP hydrolysis to translocate along single stranded nucleic acid and to unwind double stranded RNA. Here we present a detailed mechanistic analysis of the product release stage in the catalytic cycle of the dengue virus (DENV) NS3h. This study is based on a combined experimental and computational approach of product-inhibition studies and free energy calculations. Our results support a model in which the catalytic cycle of ATP hydrolysis proceeds through an ordered sequential mechanism that includes a ternary complex intermediate (NS3h-Pi-ADP), which evolves releasing the first product, phosphate (Pi), and subsequently ADP. Our results indicate that in the product release stage of the DENV NS3h a novel open-loop conformation plays an important role that may be conserved in NS3 proteins of other flaviviruses as well.

Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

Novel Lennard-Jones Parameters for Cysteine and Selenocysteine in the AMBER Force Field.

Cysteine is a common amino acid with a thiol group that plays a pivotal role in a variety of scenarios in redox biochemistry. In contrast, selenocysteine, the 21st amino acid, is only present in 25 human proteins. Classical force-field parameters for cysteine and selenocysteine are still scarce. In this context, we present a methodology to obtain Lennard-Jones parameters for cysteine and selenocysteine in different physiologically relevant oxidation and protonation states. The new force field parameters obtained in this work are available at https://github.com/MALBECC/AMBER-parameters-database. The parameters were adjusted to reproduce water radial distribution functions obtained by density functional theory ab initio molecular dynamics. We validated the results by evaluating the impact of the choice of parameters on the structure and dynamics in classical molecular dynamics simulations of representative proteins containing catalytic cysteine/selenocysteine residues. There are significant changes in protein structure and dynamics depending on the parameters choice, specifically affecting the residues close to the catalytic sites.

Acidity and nucleophilic reactivity of glutathione persulfide.

Persulfides (RSSH/RSS-) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS-), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS- presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, ßnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.

Catalysis of Peroxide Reduction by Fast Reacting Protein Thiols.

Life on Earth evolved in the presence of hydrogen peroxide, and other peroxides also emerged before and with the rise of aerobic metabolism. They were considered only as toxic byproducts for many years. Nowadays, peroxides are also regarded as metabolic products that play essential physiological cellular roles. Organisms have developed efficient mechanisms to metabolize peroxides, mostly based on two kinds of redox chemistry, catalases/peroxidases that depend on the heme prosthetic group to afford peroxide reduction and thiol-based peroxidases that support their redox activities on specialized fast reacting cysteine/selenocysteine (Cys/Sec) residues. Among the last group, glutathione peroxidases (GPxs) and peroxiredoxins (Prxs) are the most widespread and abundant families, and they are the leitmotif of this review. After presenting the properties and roles of different peroxides in biology, we discuss the chemical mechanisms of peroxide reduction by low molecular weight thiols, Prxs, GPxs, and other thiol-based peroxidases. Special attention is paid to the catalytic properties of Prxs and also to the importance and comparative outlook of the properties of Sec and its role in GPxs. To finish, we describe and discuss the current views on the activities of thiol-based peroxidases in peroxide-mediated redox signaling processes.

In Silico Insight into the Reductive Nitrosylation of Ferric Hemeproteins.

A combination of in silico methods was used to extend the experimental description of the reductive nitrosylation mechanism in ferric hemeproteins with the molecular details of the role of surrounding amino acids. The computational strategy consisted in the estimation of potential energy profiles for the transition process associated with the interactions of the coordinated N(NO) moiety with O(H2O) or O(OH-) as nucleophiles, and with distal amino acids as proton acceptors or affecting the stability of transition states. We inspected the reductive nitrosylation in three representative hemeproteins -sperm whale metmyoglobin, α subunit of human methemoglobin and nitrophorin 4 of Rhodnius prolixus. For each case, classical molecular dynamics simulations were performed in order to obtain relevant reactive conformations, and a potential energy profile for the reactive step was obtained using adiabatic mapping or nudged elastic band approaches at the QM/MM level. Specifically, we report the role of a charged Arg45 of myoglobin in destabilizing the transition state when H2O acts as nucleophile, differently to the neutral Pro43 of the hemoglobin subunit. The case of the nitrophorin is unique in that the access of the required water molecules is scarce, thus, preventing the reaction.

Spectroscopy in Complex Environments from QM-MM Simulations.

The applications of multiscale quantum-classical (QM-MM) approaches have shown an extraordinary expansion and diversification in the last couple of decades. A great proportion of these efforts have been devoted to interpreting and reproducing spectroscopic experiments in a variety of complex environments such as solutions, interfaces, and biological systems. Today, QM-MM-based computational spectroscopy methods constitute accomplished tools with refined predictive power. The present review summarizes the advances that have been made in QM-MM approaches to UV-visible, Raman, IR, NMR, electron paramagnetic resonance, and Mössbauer spectroscopies, providing in every case an introductory discussion of the corresponding methodological background. A representative number of applications are presented to illustrate the historical evolution and the state of the art of this field, highlighting the advantages and limitations of the available methodologies. Finally, we present our view of the perspectives and open challenges in the field.

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

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.

Naturally occurring fluorescence in frogs.

Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual perception in terrestrial environments.

Ligand Binding Rate Constants in Heme Proteins Using Markov State Models and Molecular Dynamics Simulations.

Computer simulation studies of the molecular basis for ligand migration in proteins allow the description of key events such as the transition between docking sites, displacement of existing ligands and solvent molecules, and open/closure of specific "gates", among others. In heme proteins, ligand migration from the solvent to the active site preludes the binding to the heme iron and triggers different functions. In this work, molecular dynamics simulations, a Markov State Model of migration and empirical kinetic equations are combined to study the migration of O2 and NO in two truncated hemoglobins of Mycobacterium tuberculosis (Mt-TrHbN and Mt-TrHbO). For Mt-TrHbN, we show that the difference in the association constant in the oxy and deoxy states relies mainly in the displacement of water molecules anchored in the distal cavity in the deoxy form. The results here provide a valuable approach to study ligand migration in globins.

Evaluation of quinoxaline compounds as ligands of a site adjacent to S2 (AS2) of cruzain.

The binding of ten quinoxaline compounds (1-10) to a site adjacent to S2 (AS2) of cruzain (CRZ) was evaluated by a protocol that include a first analysis through docking experiments followed by a second analysis using the Molecular Mechanics-Poisson-Boltzmann Surface Area method (MM-PBSA). Through them we demonstrated that quinoxaline compounds bearing substituents of different sizes at positions 3 or 4 of the heterocyclic ring might interact with the AS2, particularly interesting site for drug design. These compounds showed docking scores (ΔGdock) which were similar to those estimated for inhibitors that bind to the enzyme through non-covalent interactions. Nevertheless, the free binding energies (ΔG) values estimated by MM-PBSA indicated that the derivatives 8-10, which bear bulky substituents at position 3 of the heterocycle ring, became detached from the binding site under a dynamic study. Surprisingly, the evaluation of the inhibitory activity of cruzipain (CZ) of some derivatives showed that they increase the enzymatic activity. These results lead us to conclude about the relevance of AS2 as a pocket for compounds binding site, but not necessarily for the design of anti-chagasic compounds.

Thermal Stability of Globins: Implications of Flexibility and Heme Coordination Studied by Molecular Dynamics Simulations.

Proteins are sensitive to temperature, and abrupt changes in the normal temperature conditions can have a profound impact on both structure and function, leading to protein unfolding. However, the adaptation of certain organisms to extreme conditions raises questions about the structural features that permit the structure and function of proteins to be preserved under these adverse conditions. To gain insight into the molecular basis of protein thermostability in the globin family, we have examined three representative examples: human neuroglobin, horse heart myoglobin, and Drosophila hemoglobin, which differ in their melting temperatures and coordination states of the heme iron in the absence of external ligands. In order to elucidate the possible mechanisms that govern the thermostability of these proteins, microsecond-scale classical molecular dynamics simulations were performed at different temperatures. Structural fluctuations and essential dynamics were analyzed, indicating that the flexibility of the CD region, which includes the two short C and D helixes and the connecting CD loop, is directly related to the thermostability. We observed that a larger inherent flexibility of the protein produces higher thermostability, probably concentrating the thermal fluctuations observed at high temperature in flexible regions, preventing unfolding. Globally, the results of this work improve our understanding of thermostability in the globin family.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

The reaction of hydrogen sulfide (H2S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-FeIII) and myoglobin (Mb-FeIII), from the estimation of the intrinsic binding constants of the species H2S and hydrosulfide (HS-), and the computational description of the overall binding process. Our results show that H2S and HS- are the main reactive species in Mb-FeIII and MP11-FeIII, respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS- to Mb-FeIII was negligible, the binding of H2S to MP11-FeIII was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H2S to Mb-FeIII and that the binding of HS- is prevented in the access to the protein matrix. For the MP11-FeIII case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H2S and HS- are capable of binding a ferric heme moiety.

A quantitative model for oxygen uptake and release in a family of hemeproteins.

MOTIVATION: Hemeproteins have many diverse functions that largely depend on the rate at which they uptake or release small ligands, like oxygen. These proteins have been extensively studied using either simulations or experiments, albeit only qualitatively and one or two proteins at a time. RESULTS: We present a physical-chemical model, which uses data obtained exclusively from computer simulations, to describe the uptake and release of oxygen in a family of hemeproteins, called truncated hemoglobins (trHbs). Through a rigorous statistical analysis we demonstrate that our model successfully recaptures all the reported experimental oxygen association and dissociation kinetic rate constants, thus allowing us to establish the key factors that determine the rates at which these hemeproteins uptake and release oxygen. We found that internal tunnels as well as the distal site water molecules control ligand uptake, whereas oxygen stabilization by distal site residues controls ligand release. Because these rates largely determine the functions of these hemeproteins, these approaches will also be important tools in characterizing the trHbs members with unknown functions. CONTACT: lboechi@ic.fcen.uba.ar SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tyrosine oxidation and nitration in transmembrane peptides is connected to lipid peroxidation.

Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family.

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

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.