Reactive Oxygen and Nitrogen Species at Phospholipid Bilayers

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B: Biomaterials and Membranes

Reactive Oxygen and Nitrogen Species at Phospholipid Bilayers: Peroxynitrous Acid and Its Homolysis Products Rodrigo Maghdissian Cordeiro J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07158 • Publication Date (Web): 05 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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Reactive Oxygen and Nitrogen Species at Phospholipid Bilayers: Peroxynitrous Acid and Its Homolysis Products

Rodrigo M. Cordeiro*

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Avenida dos Estados 5001, CEP 09210-580, Santo André (SP), Brazil.

* Corresponding author. Tel.: +55 11 49960173. E-mail address: [email protected]

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Abstract

Peroxynitrite is a powerful and long-lived oxidant generated in vivo. Peroxynitrous acid (ONOOH), its protonated form, may penetrate into phospholipid bilayers and undergo homolytic cleavage to nitrogen dioxide (·NO2) and hydroxyl radicals (·OH), causing severe nitro-oxidative damage. The membrane environment is thought to influence ONOOH reactions, but the mechanisms remain speculative. Most experimental techniques lack the level of resolution required to keep track of the motion of very reactive species and their interactions with the membrane. Here, we performed molecular dynamics simulations of the permeation, interactions and dynamics of ONOOH and its homolysis products in the phospholipid membrane environment. We started by developing an ONOOH model that successfully accounted for its conformational equilibria and solvation energies. Membrane permeation of ONOOH was accompanied by conformational changes. ONOOH exhibited a strong tendency to bind to and accumulate at the membrane headgroups region. There, ONOOH homolysis led to ·NO2 radicals, which in turn partitioned to the membrane interior. About one third of the ·OH radicals readily escaped to the aqueous phase within 1 ns. However, a significant number of ·OH radicals became trapped at the lipid headgroups region for a longer period. The possible implications for membrane-based nitration and oxidation processes were discussed.


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1. Introduction

Reactive oxygen and nitrogen species (RONS) are a family of strongly oxidizing small molecules and free radicals. Examples include highly unstable oxy-radicals such as hydroxyl (·OH), hydroperoxyl (HO2·) and superoxide (·O2-), moderately stable radicals such as nitric oxide (·NO) and nitrogen dioxide (·NO2), and molecules such as peroxynitrite (ONOO−), hydrogen peroxide (H2O2) and oxygen itself (O2). Certain RONS are naturally present in the organism and participate in redox signaling pathways that are essential for the physiological control of cell function.1,2 However, excessive amounts of RONS may overwhelm the antioxidant defenses, leading to oxidative stress.3 Increased levels of RONS are associated with deleterious processes such as aging, carcinogenesis and neurodegenerative diseases.4-8 Peroxynitrite is one of the most potentially damaging RONS.8-11 It is a powerful and long-lived oxidant that is generated in vivo from the combination between nitric oxide (·NO) and superoxide radicals (·O2−). Peroxynitrite participates in one- and two-electron oxidation reactions with lipids, proteins and DNA. At physiological pH, ONOO− is in equilibrium with appreciable amounts of peroxynitrous acid (ONOOH; pKa = 6.5−6.8),11 which can undergo homolysis of the O—O bond, generating ·NO2 and the extremely reactive ·OH radical. The formation of ONOO− is at least a million times faster than the metal-catalyzed generation of ·OH from H2O2 via the Fenton reaction.12 Besides that, while ·OH radicals react only a few molecular diameters away from their generation site, ONOO− is stable enough to traverse micrometer-range distances before initiating free radical-based reactions.10,13 To a certain degree, the effects of RONS may relate to their interactions with phospholipid membranes. Reactions may be compartmentalized according to the membranecrossing ability of different RONS.14 Both ONOO− and ONOOH are able to cross phospholipid membranes, but through different routes. While ONOO− strictly depends on the



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presence of anion channels, ONOOH has the ability to passively diffuse through membranes.15 Besides that, reaction rates may change in the vicinity of or inside the membrane environment.16 In fact, intramembrane nitration of lipophilic tyrosine probes by ONOOH was shown to be more efficient than tyrosine nitration in solution.17-19 Knowing the distribution of ONOOH at the membrane-water interface may be important for the understanding its reactions in the organism, including oxidation and nitration of proteins and lipids, as well as radical scavenging by membrane antioxidants. Unfortunately, the use of molecular probes as reporters for RONS penetration is often limited by the imprecise knowledge of the probe location within the membrane. Most experimental techniques lack the level of resolution required to keep track of the motion of RONS and their interactions with the membrane. However, molecular dynamics simulations are well-suited for this task. Previous simulation studies showed that hydrophobic species such as O2 and ·NO concentrate at the membrane interior, but are not homogeneously distributed.20-22 That is because phospholipid bilayers are microheterogeneous environments with position-dependent properties such as dielectric constant and free-volume.23 Additionally, their molecular arrangement is anisotropic, meaning that solubility data based on model organic solvents are not always directly transferable to membranes.14 More recently, we have shown that H2O2 and small oxy-radicals may reside close to the phospholipid headgroups region.24 Thanks to lipid disorder, they are able to reach and interact with the unsaturations along the lipid acyl chains. That could lead to the formation of lipid oxidation products that change the membrane structure and properties.25,26 Besides that, reactive species may also translocate through aquaporin channels.27,28 Up to now, simulation studies of ONOOH have been conducted in solution,29 but not in the complex membrane environment. Here, we present molecular dynamics simulations of ONOOH and its homolysis products, ·NO2 and ·OH, at phospholipid bilayers. A model for ONOOH was developed from scratch. Previous electronic structure calculations of ONOOH have revealed a conformational ACS Paragon Plus Environment

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energy landscape with multiple local minima.30 As shown in Fig. 1a, ONOOH exists mainly in the cis-cis, cis-perp and trans-perp conformations, as defined by the O—N—O—O and N—O—O—H dihedral angles, respectively. Our model took that feature into consideration. Besides that, it was aimed at a correct description of solvation energies. After validation of the model, we evaluated the free energy of translocation of ONOOH, ·NO2 and ·OH through phospholipid bilayers. Specific interactions between RONS and the membrane were identified. We recorded the dynamics of the ·OH/·NO2 radical pair formed after ONOOH homolysis and discussed possible implications for membrane-based nitration and oxidation processes.

2. Computational methods

2.1 Simulation setup

Molecular dynamics simulations31 were performed with GROMACS 5.0.4.32,33 Newton’s equations of motion were integrated with a time step of 2 fs, with all chemical bonds constrained to their equilibrium lengths. Electrostatic interactions were treated by the particle mesh Ewald (PME) method with a real space cutoff of 0.9 nm. Lennard-Jones interactions were truncated using a twin-range cutoff at 0.9 and 1.4 nm. Long-range dispersion corrections were applied to both energy and pressure.

2.2 Force field

Interatomic interactions were based on the framework of the GROMOS 53A6 force field.34 The phospholipid 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) was described according to the interaction parameters of Poger et al.35,36 The SPC model was employed for ACS Paragon Plus Environment


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water molecules. Parameters for the ·OH radicals came from our previously developed force field for reactive oxygen species.24 In the case of ·NO2 radicals, parameters for bonds and angles were taken from the literature.37 Lennard-Jones interactions and partial charges were empirically adjusted so as to match the solvation free energy of ·NO2 in both decane38,39 and water.40,41 The Lennard-Jones parameters of ·NO2 were also designed to be transferable to dinitrogen tetroxide (N2O4) and ozone (O3).38,40-42 All solvation free energies were calculated with the thermodynamic integration method,31 following the same protocols as in ref. 24. In the case of ONOOH, parameters for bonds and angles were taken from the literature.24,30,43 The torsional potentials at the O—N—O—O and N—O—O—H fragments were adjusted to match earlier electronic structure calculations.30 Lennard-Jones parameters were taken from ·OH and ·NO2. Partial charges were developed in multiple stages. First, the geometries of representative ONOOH conformers were optimized by density functional calculations with the B3LYP functional and the 6-31G(d) basis set using Gaussian 09.44 An initial estimate of the atom-centered partial charges was obtained by fitting of the electrostatic potential.45 Charges were then iteratively scaled so as to account for solvent-induced polarization, i.e. until the model was able to reproduce the correct hydration free energy of ONOOH.11 The force field and molecular topology files of the simulated RONS are available as Supporting Information.

2.3 Permeation free energies

The free energy profiles associated to the translocation of RONS across POPC bilayers were calculated using the umbrella sampling method.46,47 The starting structures for umbrella simulations were picked randomly from the last 50 ns of a 100 ns equilibrium simulation of a fully hydrated POPC bilayer containing 128 lipids and 5941 water molecules. The system had the following dimensions: ~6.2 nm parallel to the membrane surface (xy-plane) and ~8.5 nm ACS Paragon Plus Environment

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along the bilayer normal (z-axis). Periodic boundary conditions were considered in all Cartesian directions. Evenly spaced umbrella positions were defined along the z-axis at intervals of 0.5 Å, forming complete transmembrane permeation paths. Umbrella positions were expressed in terms of center-of-mass distances between individual RONS and the bilayer. To minimize artifacts due to membrane undulation, only the bilayer atoms within a lateral distance of 1.2 nm to the permeating RONS were considered for the computation of the centers-of-mass. Permeants were attached to the umbrella positions by harmonic potentials with force constants of 1000 kJ·mol-1·nm-2 along the z-axis. Their lateral motion was confined to ~1 nm cylindrical cross sections using flat-bottomed cylindrical restraints. In each individual umbrella simulation, four independent permeation paths were sampled simultaneously, and in each of them, four spatially separated windows. There were no close contacts between individual permeants. Simulations were carried out in the isothermalisobaric (NPT) ensemble at 310 K and 1 bar. Temperature was controlled using a NoséHoover thermostat. Pressure was controlled independently along the xy-plane and the z-axis using a semiisotropic Parrinello-Rahman barostat (see ref. 24 for further details). We investigated the transmembrane permeation of ·OH, ·NO2 and ONOOH. Each umbrella window comprised 10 ns of equilibration and 10 ns of sampling. Achievement of proper convergence is an important issue in umbrella sampling simulations.48 We found that these conditions led to reasonably converged free energy profiles in the case of the small neutral permeants investigated. Umbrella histograms were collected and free energy profiles were built by the weighted histogram analysis method (WHAM).49,50 Histograms were reweighted according to an estimate of their integrated autocorrelation times. The periodicity of the free energy profile along the z-direction was considered. Statistical uncertainties were obtained by 50 repetitions of a Bayesian bootstrapping of complete histograms.

2.4 RONS distribution and dynamics of the radical pair ACS Paragon Plus Environment


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To study how RONS interact with and distribute through the membrane, a number of molecules were added to the aqueous phase of a pre-equilibrated POPC bilayer and a regular molecular dynamics simulation was run. The system contained 128 lipis, 5504 water molecules and 30 molecules of each of the radicals ·NO2 and ·OH. Besides that, there were 60 ONOOH molecules, which were initially equally divided between the conformational states with O—N—O—O cis and trans. After 20 ns of equilibration, data acquisition followed for 30 ns in the NPT ensemble at 310 K and 1 bar. The distribution of the different RONS was computed along the z-axis. A similar simulation was performed, but only with 30 ONOOH molecules at their O—N—O—O cis state. After completion, the O—O bond was cleaved. After energy minimization and 100 ps of thermalization, the trajectories of all individual ·OH/·NO2 radical pairs were followed for 50 ns. Graphical renderings of the simulated systems were done with VMD.51

3. Results

The force field parameters are summarized in Table S1. As shown in Fig. 1, the conformational and solvation properties of ONOOH and other RONS were satisfactorily described by the model. It is challenging to devise a unique classical model that accounts for the structure of all ONOOH conformers. Quantum Chemistry data have consistently revealed a moderate degree of coupling between the rotameric state of ONOOH and the length of the central N—O bond.30,52,53 That explains the small structural differences between the classical ONOOH model and reference electronic structure calculations (Fig. 1a and Table S2).30 Nevertheless, the conformational energy landscape of ONOOH was well represented by the model (Fig. 1b). The O—N—O—O region was planar and existed in either the cis or the trans conformation. In the gas phase, the cis-cis-ONOOH conformer had the lowest energy ACS Paragon Plus Environment

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due to its ring-like structure, which allowed the terminal O and H atoms to engage in an intramolecular H-bond. The out-of-plane rotation about the O—O bond disrupted this H-bond and led through a small barrier to cis-perp-ONOOH. From that state, rotation about the N—O bond conducted to trans-perp-ONOOH. That transition had a much larger barrier of ~54 kJ/mol as it passed through the perp-perp transition state. In multi-nanosecond simulations, this barrier translates into a low probability of cis-trans isomerization of the O—N—O—O region. In fact, we observed a small number of such events in our simulations, and they were properly handled during data analysis. The energy differences dictate that, at nearphysiological conditions, the cis conformation of O—N—O—O should predominate over the trans conformation. Although the dihedral potentials were fitted using cosine series with multiple terms, there were small differences between the quantum and the classical models. As stated earlier, the internal degrees of freedom in ONOOH are coupled and it is difficult to devise a unique model that perfectly matches the N—O—O—H rotation energy at both the cis and trans states of the O—N—O—O dihedral. As a side note, we mention that the dihedral fitting could be improved by developing two different conformer-specific models for ONOOH. Simulations yielded solvation free energies at infinite dilution that closely matched reference data for ONOOH and other RONS (Fig. 1c).11,38-42 Further details about the derivation of solvation free energies from experimental data are provided as Supporting Information (see eq S1 and S2). Our own electronic structure calculations revealed similar charge distributions in the cis-cis and trans-perp conformers. Therefore, a unique charge set was used for all conformers. We found that the aqueous solvation of the trans-perp conformer was slightly more favorable than cis-cis solvation by ~1 kJ/mol. The solvation structure of ONOOH resembled that of a small amphiphile.29 The strongly hydrophilic O—H group formed H-bonds with solvating waters, while the less hydrophilic O—N—O fragment led to



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the formation of a solvent cage (Figs. S1 a-c). The cis-perp conformer predominated over ciscis-ONOOH because water disrupted the intramolecular H-bond of ONOOH (Fig. S1d). Fig. 2 shows the free energy variations in transmembrane permeation events of ONOOH and its homolysis products. All energy profiles were very symmetric, which is an indication of proper convergence of the umbrella sampling simulations (see also Fig. S2 for a convergence analysis). As ONOOH approached the membrane surface from the aqueous phase, the free energy slightly decreased by ~7 kJ/mol, reaching its minimum at the phospholipid headgroups region (Figs. 2 b). As ONOOH penetrated further, the free energy steeply increased and reached a plateau in the membrane interior. The bilayer structure was not disrupted during this process, i.e. ONOOH permeation was not accompanied by the formation of a transmembrane pore (Fig. 2a). These features are consistent with the solubilitydiffusion model.23,54 According to this model, hydrophilic molecules first penetrate into the membrane, at the cost of losing hydration, and then diffuse in a nearly flat energy landscape across the membrane interior. Free energy profiles were independently recorded for cis-perpand trans-perp-ONOOH. A free energy barrier of 17 ± 2 kJ/mol was recorded for cis-perpONOOH, which is very similar to the permeation barrier of effective membrane permeants such as ethanol.22 The trans-perp conformer had a similar permeation barrier. The cis-perp conformer transitioned to cis-cis inside the membrane, forming an intramolecular H-bond (Fig. 2d). The homolysis products of ONOOH exhibited very different free energy profiles. Permeation of the ·OH radical was associated to a shallow free energy minimum at the membrane surface. The free energy then steeply increased as the ·OH radical moved to the membrane interior, where it formed a high-energy plateau. In the case of ·NO2, the permeation barrier was significantly lower and located at the headgroups region. The free energy in the membrane center was slightly lower than in the aqueous phase. Fig. 3 shows the distributions of ONOOH and its homolysis products across phospholipid bilayers, as obtained from equilibrium simulations (see also Fig. S3a for a ACS Paragon Plus Environment

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convergence analysis). The results largely corroborated the free energy profiles. Both cisperp- and trans-perp-ONOOH adsorbed and accumulated at the headgroups region, i.e. the region of the free energy minima. Adsorption was driven by the formation of H-bonds, mostly between ONOOH and the phospholipid carbonyl ester groups (Fig. S3b). The “surfactantlike” structure of ONOOH probably also had an important role. As expected, hydrophilic ·OH radicals tended to stay at the aqueous phase, but a few managed to penetrate down to the region of the carbonyl ester groups. Being only slightly polar, ·NO2 followed the tendency of small hydrophobic molecules to accumulate in the membrane interior.39 We wondered what would happen if ONOOH underwent homolysis at its membrane adsorption site. To answer this question, a POPC membrane was equilibrated in the presence of ONOOH (cis-state O—N—O—O), all ONOOH molecules were then cleaved, and the trajectories of the individual ·OH/·NO2 radical pairs were followed. These trajectories are shown in Fig. 4. The radicals separated from each other very rapidly, i.e. within less than 1 ns. The ·NO2 radicals were mostly released to the membrane interior, but some radicals escaped to the aqueous phase. Since simulations take place in a closed system, the ·NO2 radicals that managed to escape to the aqueous phase eventually re-entered the membrane. Fig. 4b shows the trajectories of individual ·NO2 molecules, up to the moment in which they managed to exit the membrane and make their first excursion to the aqueous phase. Fig. 4b suggests that, in an open system with an infinitely large aqueous phase, most ·NO2 radicals would have diffused out of the membrane within ~20 ns. In the case of ·OH, about one third of the radicals escaped to the aqueous phase within only 1 ns. However, a significant number of radicals remained bound to the headgroups region for tens of nanoseconds. Residence times reached up to ~50 ns. At first sight, this result seemed to contradict the free energy (Fig. 2b) and distribution (Fig. 3d) data, which did not reveal any particularly strong affinity between ·OH and the membrane surface. However, both sets of data are ensemble averages. A large fraction of ·OH radicals generated in situ have indeed readily escaped to the aqueous phase. ACS Paragon Plus Environment


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Long residence times were recorded for some of the ·OH radicals because they were generated in a supposedly metastable spatial configuration favoring H-bond interactions with the phospholipid carbonyl ester groups. It is conceivable that some of the ·OH radicals remained kinetically trapped in this configuration, which would otherwise not be easily accessible to exogenous radicals arriving from the aqueous phase. A second, independent simulation of the radical pair dynamics was performed, yielding similar results (Fig. S4).

4. Discussion

Intramembrane nitration of lipophilic tyrosine probes by ONOOH was shown to be more efficient than tyrosine nitration in solution.17-19 It has been hypothesized that ONOOH decomposition into ·NO2 and ·OH radicals may be favored at the membrane environment. In fact, solvation is known to affect ONOOH reactions. The standard free energy of homolysis has been estimated as 42 ± 4 kJ/mol in the gas phase and 58 ± 4 kJ/mol in aqueous solution.11 Our solvation data were well in line with these trends. If our hydration free energies at infinite dilution are converted to the standard state (see eq S2), they imply that aqueous solvation would cause an increase of ~10 kJ/mol in the standard free energy of homolysis. Based on a similar reasoning, we can speculate on the influence of the membrane environment on homolysis. The classical permeation free energy profiles of ONOOH, NO2 and ·OH can be combined in order to evaluate how differential solvation effects at the lipid headgroups region and the membrane interior influence the energetics of ONOOH homolysis. From a purely thermodynamic standpoint: ∆∆G = ∆G⋅OH + ∆G⋅ NO 2 − ∆GONOOH ,

(1)

where ∆∆G is the change in homolysis free energy due to solvent effects, and the individual ∆G terms refer to the position-dependent free energy changes of reactants and products as they permeate across the membrane. The ∆∆G profile in Fig. 2c suggests that positionACS Paragon Plus Environment

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dependent interactions in the membrane are unable to stabilize the homolysis products over the reactants. A similar conclusion is drawn if free energies are converted to the standard state. Probably, the alleged higher efficiency of intramembrane nitration by ONOOH stems from its ability to reside at the membrane-water interface region. In equilibrium simulations, the local ONOOH concentration at the membrane surface was enhanced by ~6 times with respect to the bulk aqueous concentration (Fig. 3d). As demonstrated by previous simulations, membrane fluidity and disorder causes molecules at this region to have access to oxidizable positions along the phospholipid hydrocarbon chains.24 It is also noteworthy that, when ONOOH molecules were cleaved at the headgroups region, some of the generated ·OH radicals remained bound to the membrane surface for tens of nanoseconds. ·OH radicals are extremely reactive towards organic substrates, with typical lifetimes of 1 ns.55 Our results indicate that, although some ·OH radicals may be lost to the aqueous phase right after ONOOH homolysis, a few others could remain bound to the membrane surface for long enough time to react (Fig. 4a). It has been argued that the Grotthuss-type diffusion of ·OH could become less relevant at the membrane-water interface due to its lower hydration at this region.24 That effect could also contribute to “trap” the ·OH radicals provenient from ONOOH homolysis. We emphasize that, although the total simulation time was quite short (i.e. 50 ns), simulations were meant to reflect the fast non-equilibrium dynamics of the radical pair, which also takes place in reality within a rather short time scale. The conformational state of ONOOH has a remarkably large influence on its reactivity. The cis-state of the O—N—O—O dihedral has been associated with a lower barrier for O—O bond cleavage, as compared to the trans-state.56 The membrane environment appeared not to elicit any significant stabilization of the cis over the trans isomer (Fig. 2b). However, we recall the cis-isomer is expected to largely predominate over the trans.29 Interestingly, alternative mechanisms have been proposed for ONOOH reactions that are not ACS Paragon Plus Environment


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based on the formation of physically separated ·NO2 and ·OH radical intermediates.57 It is believed that ONOOH may distort into higher energy conformations with a biradicaloid character.11,12 In fact, higher-lying singlet metastable states of ONOOH have been identified at several levels of electronic structure theory.58,59 Both cis and trans biradicaloid intermediates with elongated O—O bonds have been found in the gas phase. They were lower in energy than the H-bonded ·OH/·NO2 radical pair. Similar metastable structures have also been found in the presence of a few solvating water molecules. Purely classical models are well suited for the study of partition and permeation phenomena. However, they do not explicitly account for electronic degrees of freedom and chemical reactions. It should not be a critical issue for ONOOH, as its typical lifetimes10 largely exceed the multi-nanosecond time scale of molecular dynamics simulations. Contrarily, ·OH radicals are very reactive and classical models provide an incomplete representation their behavior. Reactivity could be accessed by hybrid quantum mechanical/molecular mechanical methods60 or even by molecular dynamics with reactive force fields.61 However, these techniques are still severely limited with regards to the time and length scales typically accessible with current computational resources. Although the N—O— O—H dihedral states have been thoroughly sampled in our work (Fig S2 c-f), conformational changes of the O—N—O—O dihedral were rare events. To circumvent this limitation, free energy profiles were reported separately for the cis and trans states of the O—N—O—O dihedral. Therefore, possible extentions of this work might include metadynamics simulations, as they have been successfully used to sample internal degrees of freedom with high energy barriers and long equilibration times.62 Here, our main achievement was to demonstrate how position-dependent interactions affect the distribution and dynamics of ONOOH and its homolysis products in phospholipid bilayers. Our results will hopefully help in the interpretation of experimental data related to oxidation and nitration reactions in the membrane environment. They may also serve as a starting point for more comprehensive ACS Paragon Plus Environment

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theoretical studies of ONOOH reactions in membranes, including the formation of the biradicaloid intermediate.

5. Conclusions

We developed a force field for ONOOH that successfully accounted for its conformational equilibria and solvation energies. The permeation of cis-perp-ONOOH through a POPC bilayer was associated with a free energy barrier of 17 ± 2 kJ/mol, being comparable to effective membrane permeants such as ethanol. In the hydrophobic membrane interior, cisperp-ONOOH transitioned to cis-cis-ONOOH, establishing an intramolecular H-bond. All ONOOH conformers had a strong tendency to bind to and accumulate at the membrane headgroups region. There, ONOOH homolysis led to ·NO2 and ·OH radicals, which had different fates. The ·NO2 radicals were mostly released to the membrane interior. One third of the ·OH radicals readily escaped for the aqueous phase within 1 ns, but some radicals remained bound to the headgroups region via H-bonds. These results cast light into previous experiments suggesting that ONOOH reactions might become more effective in the membrane environment. They will hopefully contribute to a better understanding of nitrooxidative stress in membranes.

Supporting Information

Determination of solvation free energies from experimental data (eqs S1 and S2). Force field parameters for RONS (Table S1). Structures and relative energies of ONOOH conformers (Table S2). Solvation structure and conformations of ONOOH in water (Fig. S1). Convergence of umbrella sampling simulations (Fig. S2). Convergence of equilibrium



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simulations (Fig. S3). Radical pair dynamics (Fig. S4). Force field and molecular topology files for ·OH, ·NO2, O3, N2O4 and ONOOH.

Acknowledgements

We are grateful for the financial support received from the São Paulo Research Foundation (FAPESP) (grant no. 2012/50680-5) and from the National Counsel of Technological and Scientific Development (CNPq) (grant no. 459270/2014-1). Computational resources were provided by Universidade Federal do ABC.


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Fig. 1. Conformational and solvation energies of RONS. (a) Conformers of ONOOH. Color code: white H, blue N and red O. Classifications as cis, perp and trans refer to dihedral angles near 0°, 90° and 180°, respectively. The dashed line indicates the intramolecular H-bond in cis-cis-ONOOH. (b) Relative energies of ONOOH conformers in the gas phase. The classical force field is compared to previous quantum calculations.30 (c) Simulation and experimental11,38-42 values of solvation free energies of RONS in water (w) and decane (d). The isomeric structure of the solvent was n-decane in simulations, but has not been specified in the reference experimental data.



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Fig. 2. Transmembrane permeation of ONOOH and its homolysis products. (a) Simulation image showing selected ONOOH molecules (van der Waals spheres) along their permeation path across a fully hydrated POPC membrane (lines). Color code: white H, cyan C, blue N, red O and brown P. (b) Permeation free energy profiles along the membrane normal. Data are presented for both the cis (full line) and trans (dashed line) conformations of the ONOO dihedral. Free energy differences were expressed in relation to the bulk aqueous phase and uncertainties are shown as shaded regions. (c) Contribution of the environment to the ONOOH homolysis free energy, as estimated from the combination of the free energy profiles of reactants and products (see eq 1). (d) Conformational changes of cisperp-ONOOH as it translocated across the membrane.


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Fig. 3. Simulation images highlighting the distributions of (a) ONOOH (ONOO dihedrals at the cis state), (b) ·OH and (c) ·NO2. (d) Distributions of ONOOH and its homolysis products across a POPC bilayer. The average positions of the phospholipid phosphate (z = 1.95 nm) and carbonyl ester groups (z = 1.55 nm) are indicated (vertical dashed lines). The membrane center was set at z = 0. (e) Image of an ONOOH molecule Hbonded to one of the carbonyl ester groups of POPC.



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Fig. 4. Trajectories of the (a) ·OH and (b) ·NO2 radicals generated after ONOOH homolysis. Positions are expressed in terms of the distances between radicals and the membrane center. The membrane region (grey area), as delimited by the phosphate groups, is shown along with the average position of the carbonyl ester groups (dashed line). Individual trajectories are colored according to the residence time of the respective ·OH radicals at the membrane surface. The trajectories of individual ·NO2 molecules are shown up to the moment in which they managed to exit the membrane and make their first excursion to the aqueous phase (c) Temporal evolution of the distances between ·OH and ·NO2 radicals in individual radical pairs. (d) H-bond interactions between selected ·OH radicals and phospholipid carbonyl ester groups. Colored regions indicate instants in which H-bonds existed. H-bonds were defined by donor-acceptor distances lower than 0.35 nm and donor-H-acceptor angles lower than 30°.


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Reactive Oxygen and Nitrogen Species at Phospholipid Bilayers

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