RESUMEN

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Challenging the basis of our chemical intuition, recent experimental evidence reveals the presence of a new type of intrinsic fluorescence in biomolecules that exists even in the absence of aromatic or electronically conjugated chemical compounds. The origin of this phenomenon has remained elusive so far. In the present study, we identify a mechanism underlying this new type of fluorescence in different biological aggregates. By employing non-adiabatic ab initio molecular dynamics simulations combined with a data-driven approach, we characterize the typical ultrafast non-radiative relaxation pathways active in non-fluorescent peptides. We show that the key vibrational mode for the non-radiative decay towards the ground state is the carbonyl elongation. Non-aromatic fluorescence appears to emerge from blocking this mode with strong local interactions such as hydrogen bonds. While we cannot rule out the existence of alternative non-aromatic fluorescence mechanisms in other systems, we demonstrate that this carbonyl-lock mechanism for trapping the excited state leads to the fluorescence yield increase observed experimentally, and set the stage for design principles to realize novel non-invasive biocompatible probes with applications in bioimaging, sensing, and biophotonics.

Asunto(s)

RESUMEN

The determination of minimum free energy pathways (MFEP) is one of the most widely used strategies to study reactive processes. For chemical reactions in complex environments, the combination of quantum mechanics (QM) with a molecular mechanics (MM) representation is usually necessary in a hybrid QM/MM framework. However, even within the QM/MM approximation, the affordable sampling of the phase space is, in general, quite restricted. To reduce drastically the computational cost of the simulations, several methods such as umbrella sampling require performing a priori a selection of a reaction coordinate. The quality of the computed results, in an affordable computational time, is intimately related to the reaction coordinate election which is, in general, a nontrivial task. In this work, we provide an approach to model reactive processes in complex environments that does not require the a priori selection of a reaction coordinate. The proposed methodology combines QM/MM simulations with an extrapolation of the nudged elastic bands (NEB) method to the free energy surface (FENEB). We present and apply our own FENEB scheme to optimize MFEP in different reactive processes, using QM/MM frameworks at semiempirical and density functional theory levels. Our implementation is based on performing the FENEB optimization by uncoupling the optimization of the band in a perpendicular and tangential direction. In each step, a full optimization with the spring force is performed, which guarantees that the images remain evenly distributed. The robustness of the method and the influence of sampling on the quality of the optimized MFEP and its associated free energy barrier are studied. We show that the FENEB method provides a good estimation of the reaction barrier even with relatively short simulation times, supporting that its combination with QM/MM frameworks provides an adequate tool to study chemical processes in complex environments.

RESUMEN

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.

Asunto(s)

RESUMEN

The interactions of the heme iron of hemeproteins with sulfide and disulfide compounds are of potential interest as physiological signaling processes. While the interaction with hydrogen sulfide has been described computationally and experimentally, the reaction with disulfide, and specifically the molecular mechanism for ligand binding has not been studied in detail. In this work, we study the association process for disulfane and its conjugate base disulfanide at different pH conditions. Additionally, by means of advanced sampling techniques based on multiple steered molecular dynamics, we provide free energy profiles for ligand migration for both acid/base species, showing a similar behavior to the previously reported for the related H2S/HS¯ pair. Finally, we studied the ligand interchange reaction (H2O/H2S, HS¯ and H2O/HSSH, HSS¯) by means of hybrid quantum mechanics-molecular mechanics calculations. We show that the anionic species are able to displace more efficiently the H2O bound to the iron, and that the H-bond network in the distal cavity can help the neutral species to perform the reaction. Altogether, we provide a molecular explanation for the experimental information and show that the global association process depends on a fine balance between the migration towards the active site and the ligand interchange reaction.

Asunto(s)

RESUMEN

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by inorganic disulfide species has been studied by combined spectroscopic and kinetic analyses, under argon atmosphere. The process is kinetically characterized by biexponential time traces, for variable ratios of excess disulfide to protein, in the pH interval 6.6-8.0. Using UV-vis and resonance Raman spectroscopies, we observed that MbFeIII is converted into a low spin hexacoordinated ferric complex, tentatively assigned as MbFeIII(HSS-)/MbFeIII(SS2-), in an initial fast step. The complex is slowly converted into a pentacoordinated ferrous form, assigned as MbFeII according to the resonance Raman records. The reduction is a pH-dependent process, but independent of the initial disulfide concentration, suggesting the unimolecular decomposition of the intermediate complex following a reductive homolysis. We estimated the rate of the fast formation of the complex at pH 7.4 (kon = 3.7 × 103 M-1 s-1), and a pKa2 = 7.5 for the equilibrium MbFeIII(HSS-)/MbFeIII(SS2-). Also, we estimated the rate for the slow reduction at the same pH (kred = 10-2 s-1). A reaction mechanism compliant with the experimental results is proposed. This mechanistic study provides a differential kinetic signature for the reactions of disulfide compared to sulfide species on metmyoglobin, which may be considered in other hemeprotein systems.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Persulfides (RSSH/RSS-) are species closely related to thiols (RSH/RS-) and hydrogen sulfide (H2S/HS-), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed.

RESUMEN

The reactivity of inorganic sulfide towards ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate has been explored by means of visible absorption and resonance Raman spectroscopies. The reaction has been previously studied in buffered solutions at neutral pH and in the presence of excess sulfide, revealing the formation of a moderately stable hexacoordinated low spin ferric sulfide complex that yields the ferrous form in the hour's timescale. In the surfactant solution, instead, the ferrous form is rapidly formed. The spectroscopic characterization of the heme structure in the surfactant milieu revealed the stabilization of a major ferric mono-histidyl high spin heme, which may be ascribed to out of plane distortions prompting the detachment of the axially ligated water molecule, thus leading to a differential reactivity. The ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate provides a model for pentacoordinated heme platforms with an imidazole-based ligand.

Asunto(s)

RESUMEN

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Dengue represents a substantial public health burden, particularly in low-resource countries. Non-structural protein 3 (NS3) is a multifunctional protein critical in the virus life cycle and has been identified as a promising anti-viral drug target. Despite recent crystallographic studies of the NS3 helicase domain, only subtle structural nucleotide-dependent differences have been identified, such that its coupled ATPase and helicase activities remain mechanistically unclear. Here we use molecular dynamics simulations to explore the nucleotide-dependent conformational landscape of the Dengue virus NS3 helicase and identify substantial changes in the protein flexibility during the ATP hydrolysis cycle. We relate these changes to the RNA-protein interactions and proposed translocation models for other monomeric helicases. Furthermore, we report a novel open-loop conformation with a likely escape route for Pi after hydrolysis, providing new insight into the conformational changes that underlie the ATPase activity of NS3.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Oxygen binding proteins (O2BIP) have been actively investigated for the past five decades due to their rich redox chemistry and function as O2 carriers in blood cells, as well as their function as gasotransmitters and sensors that modulate cellular signaling. A series of meetings on the periodic advances in the knowledge gained in the field of globin structure and function are conducted typically on a biannual basis. In the fall of 2018, the XXth International Conference was conducted, and very important articles with breakthrough discoveries were presented and very enthusiastically discussed. This was yet another highly successful meeting in the series. Select articles from this meeting were recently reviewed, updated, and published over several issues of Antioxidants and Redox Signaling, as Forum articles communicating the latest advances in this important area of redox biology. This Forum editorial introduces these articles and highlights their scientific significance in advancing the field. Each of these articles grew out of lectures presented in the meeting, and appears either as an original contribution or a comprehensive review in the journal. Overall, the articles published in the Forum provide in-depth details on the recent developments in the field as well as point the way to future directions. These Forum articles thus serve as an important summary of progress and the ongoing direction of this field, and serve to highlight recent advances in our understanding of O2BIP.

Asunto(s)

RESUMEN

Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical-radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO-), the product of the reaction of superoxide radical (O2•-) with •NO, but ONOO--independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.

Asunto(s)

RESUMEN

The calculation of potential energy and free-energy profiles along complex chemical reactions or rare event processes is of great interest because of their importance for many areas in chemistry, molecular biology, and material science. One typical way to generate these profiles is to add a bias potential to modify the energy surface, which can act on a selected degree of freedom in the system. However, in these cases, the quality of the result is strongly dependent on the selection of the degree of freedom over which this bias potential acts. The present work introduces a simple method for the analysis of the degree of freedom selected to describe a chemical process. The proposed methodology is based on the decomposition of contributions to the potential energy profiles by the integration of forces along a reaction path, which allows evaluating the different contributions to the energy change. This could be useful for discriminating the contributions to the energy arising from different regions of the system, which is particularly useful in systems with complex environments that must be represented using hybrid quantum mechanics/molecular mechanics schemes. Furthermore, this methodology allows in generating a quick and simple analysis of the degree of freedom which is used to describe the potential energy profile associated with the reactive process. This is computationally more accessible than the corresponding free-energy profile and can therefore be used as a simple estimator of reaction coordinate adequacy.

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)