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Darío A Estrin

Publications and source records attributed to Darío A Estrin.

18 recordsLinked to original sources
PubMed2006

Modeling heme proteins using atomistic simulations.

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.

Computer Simulation↗
PubMed2006

The catalytic mechanism of peptidylglycine alpha-hydroxylating monooxygenase investigated by computer simulation.

The molecular basis of the hydroxylation reaction of the Calpha of a C-terminal glycine catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) was investigated using hybrid quantum-classical (QM-MM) computational techniques. We have identified the most reactive oxygenated species and presented new insights into the hydrogen abstraction (H-abstraction) mechanism operative in PHM. Our results suggest that O(2) binds to Cu(B) to generate Cu(B)(II)-O(2)(.-) followed by electron transfer (ET) from Cu(A) to form Cu(B)(I)-O(2)(.-). The computed potential energy profiles for the H-abstraction reaction for Cu(B)(II)-O(2)(.-), Cu(B)(I)-O(2)(.-), and [Cu(B)(II)-OOH](+) species indicate that none of these species can be responsible for abstraction. However, the latter species can spontaneously form [Cu(B)O](+2) (which consists of a two-unpaired-electrons [Cu(B)O](+) moiety ferromagnetically coupled with a radical cation located over the three Cu(B) ligands, in the quartet spin ground state) by abstracting a proton from the surrounding solvent. Both this monooxygenated species and the one obtained by reduction with ascorbate, [Cu(B)O](+), were found to be capable of carrying out the H-abstraction; however, whereas the former abstracts the hydrogen atom concertedly with almost no activation energy, the later forms an intermediate that continues the reaction by a rebinding step. We propose that the active species in H-abstraction in PHM is probably [Cu(B)O](+2) because it is formed exothermically and can concertedly abstract the substrate HA atom with the lower overall activation energy. Interestingly, this species resembles the active oxidant in cytochrome P450 enzymes, Compound I, suggesting that both PHM and cytochrome P450 enzymes may carry out substrate hydroxylation by using a similar mechanism.

Catalysis↗
PubMed2006

Heme protein oxygen affinity regulation exerted by proximal effects.

Heme proteins are found in all living organisms and are capable of performing a wide variety of tasks, requiring in many cases the binding of diatomic ligands, namely, O(2), CO, and/or NO. Therefore, subtle regulation of these diatomic ligands' affinity is one of the key issues for determining a heme protein's function. This regulation is achieved through direct H-bond interactions between the bound ligand and the protein, and by subtle tuning of the intrinsic heme group reactivity. In this work, we present an investigation of the proximal regulation of oxygen affinity in Fe(II) histidine coordinated heme proteins by means of computer simulation. Density functional theory calculations on heme model systems are used to analyze three proximal effects: charge donation, rotational position, and distance to the heme porphyrin plane of the proximal histidine. In addition, hybrid quantum-classical (QM-MM) calculations were performed in two representative proteins: myoglobin and leghemoglobin. Our results show that all three effects are capable of tuning the Fe-O(2) bond strength in a cooperative way, consistently with the experimental data on oxygen affinity. The proximal effects described herein could operate in a large variety of O(2)-binding heme proteins-in combination with distal effects-and are essential to understand the factors determining a heme protein's O(2) affinity.

Binding Sites↗
PubMed2006

Ligand-induced dynamical regulation of NO conversion in Mycobacterium tuberculosis truncated hemoglobin-N.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of "truncated hemoglobin" N (trHbN), formally acting as NO-dioxygenase, yielding the harmless nitrate ion. X-ray crystal structures have shown that trHbN hosts a two-branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate-limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 micros), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy-trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual-path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification.

Binding Sites↗
PubMed2006

A surprisingly stable S-nitrosothiol complex.

In this work we present for the first time an X-ray structure of a coordinated S-nitrosothiol obtained by reaction of the extremely reactive K[IrCl5NO] with benzylmercaptan in acetonitrile. This surprisingly stable compound, trans-K[IrCl4(CH3CN)N(O)SCH2Ph], was isolated in high yield (80%) and fully characterized by FTIR, 1H NMR, and ESI-MS. To our knowledge this is the first example of a coordinated S-nitrosothiol that has been isolated.

Journal Article↗
PubMed2005

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study.

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 x Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe(+2)) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe-S bond and yields five coordinate complexes. In the ferric (Fe(+3)) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe(+3)-NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe(+2)-NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.

Binding Sites↗
PubMed2005

Solvent effects on peroxynitrite structure and properties from QM/MM simulations.

We have investigated the structure and the vibrational spectrum of peroxynitrite anion in aqueous solution by means of combined quantum-classical (QM/MM) molecular dynamics simulations. In our QM/MM scheme, the reactant was modeled using density functional theory with a Gaussian basis set and the solvent was described using the mean-field TIP4P and the polarizable TIP4P-FQ force fields. The choice of basis sets, functionals and force field parameters has been validated by performing calculations on isolated peroxynitrite and on small peroxynitrite-water complexes. Poor values for isolated peroxynitrite structural properties and vibrational frequencies are found for most ab initio methods, particularly regarding the ON-OO(-) bond distance and the harmonic stretching frequency, probably due to the singlet-triplet instability found in the HF wave function. On the other hand, DFT methods yield results in better agreement with high level CCSD(T) ab initio calculations. We have computed the vibrational spectrum for aqueous peroxynitrite by calculating the Fourier transform of the velocity autocorrelation function extracted from the QM-MM molecular dynamics simulations. Our computational scheme, which allows for the inclusion of both anharmonicity and solvent effects, is able to clarify previous discrepancies regarding the experimental spectra assignments and to shed light on the subtle interplay between solvation and peroxynitrite structure and properties.

Computer Simulation↗
PubMed2005

Solvation and structure of LiAlH(4) in ethereal solvents.

The nature of the solute species present in ethereal solutions of LiAlH(4) is of crucial importance for understanding the mechanisms for the reduction of ketones and other functional groups by LiAlH(4). We have employed a combination of theoretical and experimental techniques to investigate the structure of LiAlH(4) in ethereal solutions. Using complexation agents, we measured the IR spectra of LiAlH(4) and AlH(4)(-) in tetrahydrofuran (THF). Hybrid quantum-classical (QM-MM) simulations have also been carried out to compute the IR spectra of associated and dissociated LiAlH(4) species and the free-energy profile for the dissociation process in solution. Our experimental estimate of the dissociation constant in THF is 0.021 +/- 0.002, while the predicted computational value corresponding to a model dimethyl ether solvent is 0.001. The free-energy profile shows only one minimum corresponding to a contact ion pair at a Li-Al separation distance of 3.0 A. These results are consistent with the fact that LiAlH(4) is essentially associated in ethereal solutions forming contact ion pairs.

Aluminum Compounds↗
PubMed2005

Mechanisms of NO release by N1-nitrosomelatonin: nucleophilic attack versus reducing pathways.

A new type of physiologically relevant nitrosamines have been recently recognized, the N(1)-nitrosoindoles. The possible pathways by which N(1)-nitrosomelatonin (NOMel) can react in physiological environments have been studied. Our results show that NOMel slowly decomposes spontaneously in aqueous solution, generating melatonin as the main organic product (k = (3.7 +/- 1.1) x 10(-5) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C, anaerobic). This rate is accelerated by acidification (k(pH 5.8) = (4.5 +/- 0.7) x 10(-4) s(-1), k(pH 8.8) = (3.9 +/- 0.6) x 10(-6) s(-1), Tris-HCl (0.2 M) buffer at 37 degrees C), by the presence of O(2) (k(o) = (9.8 +/- 0.1) x 10(-5) s(-1), pH 7.4, 37 degrees C, [NOMel] = 0.1 mM, P(O(2)) = 1 atm), and by the presence of the spin trap TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl; k(o) = (2.0 +/- 0.1) x 10(-4) s(-1), pH 7.4, 37 degrees C, [NOMel] = 0.1 mM, [TEMPO] = 9 mM). We also found that NOMel can transnitrosate to l-cysteinate, producing S-nitrosocysteine and melatonin (k = 0.127 +/- 0.002 M(-1) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C). The reaction of NOMel with ascorbic acid as a reducing agent has also been studied. This rapid reaction produces nitric oxide and melatonin. The saturation of the observed rate constant (k = (1.08 +/- 0.04) x 10(-3) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C) at high ascorbic acid concentration (100-fold with respect to NOMel) and the pH independence of this reaction in the pH range 7-9 indicate that the reactive species are ascorbate and melatonyl radical originated from the reversible homolysis of NOMel. Taking into account kinetic and DFT calculation data, a comprehensive mechanism for the denitrosation of NOMel is proposed. On the basis of our kinetics results, we conclude that under physiological conditions NOMel mainly reacts with endogenous reducing agents (such as ascorbic acid), producing nitric oxide and melatonin.

Ascorbic Acid↗
PubMed2005

Multiple-steering QM-MM calculation of the free energy profile in chorismate mutase.

A novel technique for computing free energy profiles in enzymatic reactions using the multiple steering molecular dynamics approach in the context of an efficient QM-MM density functional scheme is presented. The conversion reaction of chorismate to prephenate catalyzed by the Bacillus subtilis enzyme chorismate mutase has been chosen as an illustrative example.

Chorismate Mutase↗
PubMed2005

Discrimination of nitroxyl and nitric oxide by water-soluble Mn(III) porphyrins.

The water-soluble manganese(III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (Mn(III)TEPyP) and manganese(III) meso-(tetrakis(4-sulfonato-phenyl)) porphyrinate (Mn(III)TPPS) are able to chemically distinguish between HNO and NO donors, reacting with the former in a fast, efficient, and selective manner with concomitant formation of the {MnNO}(7) complex (k(on(HNO)) approximately equal to 10(5) M(-1) s(-1)), while they are inert or react very slowly with NO donors. DFT calculations and kinetic data suggest that HNO trapping is operative at least in the case of Mn(III)TPPS, while catalytic decomposition of the HNO donors (sodium trioxodinitrate and toluene sulfohydroxamic acid) seems to be the main pathway for Mn(III)TEPyP. In the presence of oxygen, the product Mn(II)TEPyP(NO) oxidizes back to Mn(III)TEPyP, making it possible to process large ratios of nitroxyl donor with small amounts of porphyrin.

Anaerobiosis↗
PubMed2005

Theoretical study of the truncated hemoglobin HbN: exploring the molecular basis of the NO detoxification mechanism.

Mycobacterium tuberculosis is the causative agent of human tuberculosis. The nitric oxide reaction with oxy-truncated hemoglobin N (trHbN) has been proposed to be responsible for the resistance mechanism by which this microorganism can evade the toxic effects of NO. In this work, we explore the molecular basis of the NO detoxification mechanism using a combination of classical and hybrid quantum-classical (QM-MM) simulation techniques. We have investigated the structural flexibility of the protein, the ligand affinity properties, and the nitric oxide reaction with coordinated O2. The analysis of the classical MD trajectory allowed us to identify Phe62 as the gate of the main channel for ligand diffusion to the active site. Moreover, the opening of the channel stems from the interplay between collective backbone motions and local rearrangements in the side chains of the residues that form the bottleneck of the tunnel. Even though the protein environment is not found to make a significant contribution to the heme moiety catalyzed reaction, the binding site influences the physiological function of the enzyme at three different levels. First, by isolating the intermediates formed in the reaction, it prevents nondesired reactions from proceeding. Second, it modulates the ligand (O2, NO) affinity of the protein, which can be ascribed to both distal and proximal effects. Finally, the stabilization of the Tyr33-Gln58 pair upon O2 binding might alter the essential dynamics of the protein, leading in turn to a mechanism for ligand-induced regulation.

Heme↗
PubMed2004

Ab initio study of NMR 15N chemical shift differences induced by Ca2+ binding to EF-hand proteins.

EF-hands are Ca(2+) binding motifs that are widely distributed throughout the entire living organism kingdom. At present, relatively little is known at a quantum mechanical level about the mechanisms that allow Ca(2+) to be recognized specifically by EF-hands and to induce a conformational switch from a compact ("closed") conformation to an "open" state that exposes a large patch of hydrophobic residues. Here, we present a study of NMR (15)N chemical shifts based on ab initio quantum mechanical calculations carried out on a minimalist model system linking both Ca(2+) binding sites across the beta-sheet of an EF-hand domain. Calculated and experimentally determined chemical shift changes are correlated with structural changes induced upon metal binding. The effect of Ca(2+) binding on these (15)N shifts can be dissected into two main contributions: one from pi-polarization of beta-sheet amide groups and the other from rotation of an isoleucine side chain. By correlating this description with experimental evidence, different polarization states for the beta-sheet amide groups were identified and linked to the overall conformation of different EF-hand domains. When all four beta-sheet amide groups are polarized, the ab initio calculations in our model indicate a cooperative stabilization effect due to the establishment of a circular network of donor-acceptor interactions connecting the two Ca(2+) ions across the beta-sheet. The emerging hypothesis from our analysis is that this cooperative network of interactions is essential for stabilizing the "open" conformation of an EF-hand domain.

Amino Acid Motifs↗
PubMed2004

NMR and molecular dynamics studies of the interaction of melatonin with calmodulin.

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca(2+) concentration via activation of its G-protein-coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring (15)N and (1)H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca(2+) by paramagnetic Tb(3+) allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.

Binding Sites↗
PubMed2003

Fast nitroxyl trapping by ferric porphyrins.

Iron(II) porphyrin nitrosyl complexes are obtained in high yields from the reaction of iron(III) porphyrins with the nitroxyl donors sodium trioxodinitrate and toluensulfohydroxamic acid. The reaction was found to proceed both in organic solvents and in aqueous media from iron(III) (meso-tetraphenyl) porphyrinate ([FeIII(TPP)]+) and iron(III) meso-tetrakis (4-sulfonatophenyl) porphyrinate ([FeIII(TPPS)]3-) or iron(III) protoporphyrin IX, respectively. The kinetic rate constant for the reaction of ([FeIII(TPPS)]3-) with sodium trioxodinitrate (kon) was estimated to be 1.00 +/- 0.04 x 107 M-1 s-1. As well as resulting in a versatile method for obtaining ferrous nitrosyl porphyrins, the reaction points at ferric porphyrins as efficient nitroxyl traps and provides a tool to model nitroxyl reactivity toward hemeproteins.

Ferric Compounds↗
PubMed2003

Modulation of the NO trans effect in heme proteins: implications for the activation of soluble guanylate cyclase.

The binding of NO to the iron heme in guanylate cyclase and other heme proteins induces the cleavage of the proximal histidine bonded to the metal. In this study we assess by means of density functional theory (DFT) electronic structure calculations the role of H-bonding to histidine in the modulation of this effect. We have considered in the first place a model of the isolated active site coordinated with imidazole and imidazolate to mimic the effects of a very strong H-bond. We have also investigated four selected ferrous heme proteins with different proximal histidine environments: the O(2) sensing FixL, horseradish peroxidase C, and the alpha and beta subunits of human hemoglobin. Our results indicate that polarization and charge transfer effects associated with H-bonding to the proximal histidine play a fundamental role in the modulation of the NO trans effect in heme proteins. We also find computational evidence suggesting that protein structural constraints may affect significantly the cleavage of the Fe-His bond.

Binding Sites↗
PubMed2002

Physiological concentrations of melatonin inhibit the nitridergic pathway in the Syrian hamster retina.

In the present work, the effect of melatonin on the hamster retinal nitridergic pathway was examined. When the retinas were incubated in the presence of low concentrations (1 pM-10 nM) of melatonin for 15 min, a significant decrease of nitric oxide synthase (NOS) activity was observed. However, when crude retinal homogenates were preincubated with melatonin for 15 min, no changes in NOS activity were detected, despite the fact that under the same conditions trifluoperazine, a calmodulin inhibitor, significantly decreased enzymatic activity. Kinetic analysis showed that melatonin decreased the V(max) of retinal NOS without changes in the K(m). On the other hand, low concentrations (100 pM) of melatonin significantly reduced retinal L-arginine influx. A decrease in the V(max) of L-arginine uptake was observed in the presence of melatonin, whereas the K(m) remained unchanged. Melatonin significantly inhibited the accumulation of cyclic guanosine monophosphate (cGMP) levels induced by both L-arginine and sodium nitroprusside (SNP). In summary, the present results indicate that melatonin could be a potent inhibitor of the retinal nitridergic pathway.

Animals↗