Understanding the EPR parameters of glycine-derived radicals: the case of N-acetylglycyl in the N-acetylglycine single-crystal environment

As a step toward an in-depth understanding of the electron paramagnetic resonance parameters of glycyl radicals in proteins, the hyperfine tensors and, particularly, the g-tensor of N-acetylglcyl in the environment of a single crystal of N-acetylglycine have been studied by systematic state-of-the-art quantum chemical calculations on various suitable model systems. The quantitative computation of the g-tensors for such glycyl-derived radicals is a veritable challenge, mainly because of the very small g-anisotropy combined with a nonsymmetrical, delocalized spin-density distribution and several atoms with comparable spin-orbit contributions to the g-tensors. The choice of gauge origin of the magnetic vector potential, and of approximate spin-orbit operators, both turn out to be more critical than found in previous studies of g-tensors for organic radicals. Environmental effects, included by supermolecular hydrogen-bonded models, were found to be moderate, because of a partial compensation between the influences from intramolecular and intermolecular hydrogen bonds. The largest effects on the g-tensor are caused by the conformation of the radical. The density functional theory methods employed systematically overestimate both the Delta gx and Delta gy components of the g-tensor. This is important for parallel investigations on the protein-glycyl radicals. The 1H alpha and 13C alpha hyperfine couplings depend only slightly on the supermolecular model chosen and appear less sensitive probes of detailed structure and environment.     

EPR parameters of amino acid radicals in P. eryngii versatile peroxidase and its W164Y variant computed at the QM/MM level

Quantum mechanics/molecular mechanics (QM/MM) methods, employing density functional theory (DFT), have been used to compute the electron paramagnetic resonance (EPR) parameters of tryptophan and tyrosyl radical intermediates involved in the catalytic cycle of Pleurotus eryngii versatile peroxidase (VP) and its W164Y variant, respectively. These radicals have been previously experimentally detected and characterized both in the two-electron and one-electron activated forms of the enzymes. In this work, the well-studied W164 radical in VP has been chosen for calibration purposes because its spectroscopic properties have been extensively studied by multifrequency EPR and ENDOR spectroscopies. Using a B3LYP/CHARMM procedure, appropriately accounting for electrostatic, such as hydrogen bonding, and steric environmental interactions, a good agreement between the calculated and measured EPR parameters for both radicals has been achieved; g-tensors, hyperfine coupling constants (hfcc) and Mulliken spin densities have been correlated to changes in geometries, hydrogen bond networks and electrostatic environment, with the aim of understanding the influence of the protein surroundings on EPR properties. In addition, the present calculations demonstrate, for VP, the formation of a neutral tryptophan radical, hydrogen bonded to the nearby E243, via a stepwise electron and proton transfer with earlier involvement of a short-lived tryptophan cationic species. Instead, for W164Y, the QM/MM dynamics simulation shows that the tyrosine oxidation proceeds via a concerted electron and proton transfer and is accompanied by a significant reorganization of residues and water molecules surrounding the tyrosyl radical.     

Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their β-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.     

Complete structural and magnetic characterization of biological radicals in solution by an integrated quantum mechanical approach: glycyl radical as a case study

An integrated quantum mechanical approach for the structural and magnetic characterization of flexible free radicals in solution has been applied to a model of the glycyl radical engaged in peptidic chains. The hyperfine couplings computed using hybrid density functionals and purposely tailored basis sets are in good agreement with experiment when vibrational averaging effects from low frequency motions and solvent effects (both direct H bonding and bulk) are taken into the proper account. The g tensor shows a smaller dependence on the specific form of the density functional, the extension of the basis set over a standard double-zeta+polarization level, vibrational averaging, and bulk solvent effects. However, hydrogen bridges with solvent molecules belonging to the first solvation shell play a significant role. Together with their intrinsic interest, our results show that a comprehensive and reliable computational approach is becoming available for the complete characterization of open-shell systems of biological interest in their natural environment.     

Protein-cofactor interactions and EPR parameters for the Q(H) quinone binding site of quinol oxidase. A density functional study

Recent multifrequency EPR studies of the "high-affinity" quinone binding site of quinol oxidase (Q(H) site) have suggested a very asymmetric hydrogen-bonding environment for the semiquinone radical anion state. Single-sided hydrogen bonding to the O1 carbonyl position was one of the proposals, which contrasts with some previous experimental indications. Here density functional calculations of the EPR parameters (g-tensors, 13C, 1H, and 17O hyperfine tensors) for a wide variety of supermolecular model complexes have been used to provide insight into the detailed relations among structure, environment, and EPR parameters of ubisemiquinone radical anions. A single-sided binding model is not able to account for the experimentally observed low g(x) component of the g-tensor or for the observed magnitude of the asymmetry of the 13C carbonyl HFC tensors. Based on the detailed comparison between computation and experiment, a model with two hydrogen bonds to O1 and one hydrogen bond to O4 is suggested for the Q(H) site, but a model with one more hydrogen bond on each side cannot be excluded. Several general conclusions on the interrelations between EPR parameters and hydrogen bond patterns of ubisemiquinones in proteins are provided.     

Pulse EPR and ENDOR study of 1,2,3-benzodithiazolyl, 2,1,3-benzothiaselenazolyl and 1,2,3-benzodiselenazolyl radicals

1,2,3-Benzodithiazolyl, 2,1,3-benzothiaselenazolyl and 1,2,3-benzodiselenazolyl radicals were generated by the reduction of the corresponding cations and investigated by pulse EPR and ENDOR in frozen CHCl(3) solutions at 30 and 80 K. These methods, in combination with density functional theory calculations, were used to study the magnetic parameters of the radicals, namely the principal values of the nitrogen and proton hyperfine interactions and g-tensors. The spin density distribution was shown to be nearly the same for all investigated radicals and, therefore, replacement of sulfur by selenium leads to a limited perturbation of the radicals' electronic structure. A high anisotropy of the g-tensors was found for the selenium-containing radicals.     

Unraveling the role of stereo-electronic, dynamical, and environmental effects in tuning the structure and magnetic properties of glycine radical in aqueous solution at different pH values

A recently developed extended Lagrangian model employing localized basis functions and nonperiodic boundary conditions (GLOB/ADMP) was applied to the radicals issuing from the homolytic breaking of the C(alpha)-H(alpha) bond of glycine in aqueous solution at different pH values. Although the modifications of the structure and the magnetic properties of these species induced by the solvent are qualitatively reproduced by a static discrete-continuum model, magnetic parameters are further tuned by short-time dynamical effects (solute vibrations and solvent librations). The results delivered by GLOB/ADMP simulations for both hyperfine tensors and g-tensors are in remarkable agreement with their experimental counterparts, allowing a reliable disentanglement of the overall observables into well-defined contributions. The dominant role of out-of-plane vibrations in determining hyperfine splittings is confirmed and quantified, together with the remarkable sensitivity of the gyromagnetic tensor to bond lengths and valence angles defining the NC(alpha)C' moiety. Together with their specific interest for the title radical, our results suggest some interesting trends for other biologically significant radicals and point out the need of extending magneto-structural relationships to dynamical aspects.     

Combined electron magnetic resonance and density functional theory study of 10 K X-irradiated beta-D-fructose single crystals

Primary free radical formations in fructose single crystals X-irradiated at 10 K were investigated at the same temperature using X-band Electron Paramagnetic Resonance (EPR), Electron Nuclear Double Resonance (ENDOR) and ENDOR induced EPR (EIE) techniques. ENDOR angular variations in the three principal crystallographic planes and a fourth skewed plane allowed the unambiguous determination of five proton hyperfine coupling tensors. From the EIE studies, these hyperfine interactions were assigned to three different radicals, labeled T1, T1* and T2. For the T1 and T1* radicals, the close similarity in hyperfine coupling tensors suggests that they are due to the same type of radical stabilized in two slightly different geometrical conformations. Periodic density functional theory calculations were used to aid the identification of the structure of the radiation-induced radicals. For the T1/T1* radicals a C3 centered hydroxyalkyl radical model formed by a net H abstraction is proposed. The T2 radical is proposed to be a C5 centered hydroxyalkyl radical, formed by a net hydrogen abstraction. For both radicals, a very good agreement between calculated and experimental hyperfine coupling tensors was obtained.     

Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory

1H hyperfine (hf) coupling constants of semiquinone radical anions of 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, and 2-methyl-3-phytyl-1,4-naphthoquinone in frozen alcoholic solutions were measured using pulse Q-band electron nuclear double resonance spectroscopy. The resolved signals of the quinone protons as well as from hydrogen bond and solvent shell protons were analyzed and assigned. Both in-plane and out-of-plane hydrogen bonding with respect to the pi-plane of the radical is observed. Interactions with nonexchangeable protons from the surrounding matrix are detected and assigned to solvent protons above and below the quinone plane. Density functional theory was used to calculate spin Hamiltonian parameters of the radical anions. Solvent molecules of the first solvent shell that provide hydrogen bonds to the quinones were included in the geometry optimization. The conductor-like screening model was employed to introduce additional effects of the solvent cage. From a comparison of the experimental and calculated hf tensors it is concluded that four solvent molecules are coordinated via hydrogen bonds to the quinone oxygens. For all radicals very good agreement between experimental and calculated data is observed. The influence of different substituents on the spin density distribution and hydrogen bond geometries is discussed.     

Theoretical studies of alkyl radicals in the NaY and HY zeolites

Interplay of quantum mechanical calculations and experimental data on hyperfine coupling constants of ethyl radical in zeolites at several temperatures was engaged to study the geometries and binding energies and to predict the temperature dependence of hyperfine splitting of a series of alkyl radicals in zeolites for the first time. The main focus is on the hyperfine interaction of alkyl radicals in the NaY and HY zeolites. The hyperfine splitting for neutral free radicals and free radical cations is predicted for different zeolite environments. This information can be used to establish the nature of the muoniated alkyl radicals in the NaY and HY zeolites via muSR experiments. The muon hyperfine coupling constants of the ethane radical cation in these zeolites are very large with relatively little dependence on temperature. It was found that the intramolecular dynamics of alkyl free radicals are only weakly affected by their strong binding to zeolites. In contrast, the substrate binding has a significant effect on their intermolecular dynamics.     

Radiation-induced radicals in glucose-1-phosphate. I. Electron paramagnetic resonance and electron nuclear double resonance analysis of in situ X-irradiated single crystals at 77 K

Electron magnetic resonance analysis of radiation-induced defects in dipotassium glucose-1-phosphate dihydrate single crystals in situ X-irradiated and measured at 77 K shows that at least seven different carbon-centered radical species are trapped. Four of these (R1-R4) can be fully or partly characterized in terms of proton hyperfine coupling tensors. The dominant radical (R2) is identified as a C1-centered species, assumedly formed by a scission of the sugar-phosphate junction and the concerted formation of a carbonyl group at the neighboring C2 carbon. This structure is chemically identical to a radical recently identified in irradiated sucrose single crystals. Radical species R1 and R4 most likely are C3- and C6-centered species, respectively, both formed by a net hydrogen abstraction. R3 is suggested to be chemically similar to but geometrically different from R4. Knowledge of the identity of the sugar radicals present at 77 K provides a first step in elucidating the formation mechanism of the phosphoryl radicals previously detected after X-irradiation at 280 K. In paper II, the chemical identity, precise conformation, and possible formation mechanisms of these radical species are investigated by means of DFT calculations and elementary insight into the radiation chemistry of sugar and sugar derivatives is obtained.     

Substituent effect on g-tensor: multifrequency ESR study and DFT calculation of polycrystalline phenoxyl radicals in diamagnetic crystals

The substituent effect on the g-tensor of polycrystalline 2,6-di-tert-butyl phenoxyl radical derivatives diluted in diamagnetic crystals was investigated using multifrequency ESR spectroscopy and DFT calculations. It was revealed that the g-tensors of the series of phenoxyl radical derivatives essentially have an orthorhombic symmetry. For some radicals, the hyperfine-splitting tensors from the para groups were resolved. The interpretations and the assignments of the spin-Hamiltonian parameters were confirmed with computer simulations in all bands. The DFT-calculated g-tensors were consistent with the experimental g-tensors. Furthermore, the shifts Delta(g) from the free electron ge were analyzed in details as the sum of three contributions. The spin-orbit interactions were found to be the dominant factor with regard to the Delta(g). With a focus on the s-o term, thus, the relationship of the g-values and the electronic excited states was explained by visualizing the molecular orbitals of the phenoxyl radical derivatives. This study thus showed the very significant potential of the combination of a multi-frequency ESR approach and a DFT calculation to advanced ESR analysis, particularly, g-tensor analysis, even for a powder-sample radical.     

EPR and ENDOR study of radiation-induced radical formation in purines: hypoxanthine hydrochloride monohydrate crystals X-irradiated at 10 K

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) study of hypoxanthine.HCl.H(2)O crystals irradiated at low temperatures (10 K) identified three radical species. In these crystals, the parent molecules exist in a cationic form with a proton at N7. R1 was the product of net hydrogen addition to N3 and exhibited alpha-proton hyperfine couplings to HC2, HN1, HC8, and HN3. The coupling to HC2 has an isotropic component smaller than usual, evidently an indication that the bonds to C2 are nonplanar. R2 was the product of net hydrogen loss from N7, equivalent to the one-electron oxidation product of neutral hypoxanthine, and exhibited alpha-proton hyperfine couplings to HC2 and HC8. Both couplings are characteristic of planar bonding arrangements at the centers of spin. R3 was provisionally identified as the product of net hydrogen addition to O6 and exhibited hyperfine alpha-proton couplings to HC8 and NH1. To identify the set of radicals, the experiments employed four crystal types: normal, deuterated only at NH positions, deuterated at HC8 and NH positions, and deuterated at HC8 only. The low-temperature data also showed clear evidence for H/D isotope effects in formation and/or stabilization of all radicals. To aid and support the identifications, the experimental results were compared to DFT calculations performed on a variety of radical structures plausible for the parent molecule and molecular packing within the crystal.     

Conformational disorder in the propagating radical of dimethacrylate polymers

Photopolymerization of methacrylic monomers yields samples with trapped radicals that are easily detected by electron paramagnetic resonance (EPR) spectroscopy. Despite its simplicity, there is no general agreement about the interpretation of this spectrum, in particular, about the role of methylene β protons. An extensive ENDOR study of the propagating radical in photopolymerized dimethacrylates has been carried out in order to obtain detailed information about methylene hyperfine couplings and, thus, about radical conformation. It is shown that literature models are not able to reproduce the ENDOR results and that only accurate fitting of ENDOR spectra obtained by saturating the EPR spectrum at different positions gives reliable information about radical conformation, thanks to the exploitation of conformational selectivity. It turns out that most radicals are in the minimum energy conformation, but any possible conformation is assumed by non negligible fractions of radical.     

Structures,g-tensors,and hyperfine coupling constants of L-α-alanine radicals in radiation dosimetry: An ab initio molecular dynamics simulation study

Alanine is used as a transfer standard dosimeter for gamma ray and electron beam calibration. An important factor affecting its dosimetric response is humidity which can lead to errors in absorbed dose calculations. Ab initio molecular dynamics calculations were performed to determine the environmental effects on the electron paramagnetic resonance (EPR) parameters of L-α-alanine radicals in acidic and alkaline solutions. A new result, not dissimilar to the closed-shell amino acid molecule alanine, is that the non-zwitterionic form of the alanine radical is the stable form in the gas phase while the zwitterionic neutral alanine radical is not a stable structure in the gas phase. Geometric and EPR parameters of radicals in both gas and solution phases are found to be dependent on hydrogen bonding of water molecules with the polar groups and on dynamic solvation. Calculations on the optimized free radicals in the gas phase revealed that for the neutral radical, hydrogen bonding to water molecules drives a decrease in the magnitudes of g-tensor components g _xx and g _yy without affecting neither g _zz component nor the hyperfine coupling constants (HFCCs). The transfer from the gas to solution phase of the alanine radical anion is accompanied with an increase in the spin density on the carboxylic group's oxygen atoms. However, for the neutral radical, this transfer from gas to solution phase is accompanied with the decrease in the spin density on oxygen atoms. Calculated isotropic HFCCs and g-tensor of all radicals are in good agreement with experiment in both acidic and alkaline solutions.     

Environmental effects on phenoxyl free radical spin densities and hyperfine couplings

The effect of the environment, as modelled by hydrogen bonding, ion-pairing and/or general continuum model effects, is investigated for the phenoxyl free radical. All are shown to lead to a redistribution of spin density from the phenoxyl O atom to the C_ipso position. Isotropic and anisotropic hyperfine couplings are calculated at the B3LYP level of theory confirming this trend. Introduction of the continuum model has a significant effect the Na-O bond length of the ion-pair model significantly altering its calculated EPR properties in comparison with gas-phase values. The trends identified are of immediate significance for biological environmental effects on tyrosyl free radicals.     

Very high-field EPR study of glycyl radical enzymes

This work shows that very high-field EPR spectroscopy allows a rather accurate determination of the g-tensor of protein radicals, including C-centered ones, and thus may be used as a probe for distinguishing a tyrosyl-, a glycyl-, or a tryptophanyl-radical. In this paper, we report the first complete analysis of the g-tensor of glycyl radical enzymes (anaerobic ribonucleotide reductase, pyruvate formate lyase, and benzylsuccinate synthase), thus providing new information on their EPR properties. Because the g-anisotropy is small, the complete resolution of the g-tensor could be only obtained at very high field (18.8 T).     

Density functional calculations of electronic g-tensors for semiquinone radical anions. The role of hydrogen bonding and substituent effects

A recently developed density functional approach has been used to carry out a systematic computational study of electronic g-tensors for a series of 1,4-semiquinone radical anions. Good agreement with high-field EPR data in frozen 2-propanol is achieved only after taking into account the significant reduction of g-tensor anisotropy caused by hydrogen bonding to solvent molecules. The comparison of various model systems for the first solvation shell suggests two hydrogen bonds from 2-propanol molecules to each of the carbonyl groups of the radical anions, and one additional hydrogen bond to each of the methoxy groups in ubiquinone systems. 2-Propanol makes stronger hydrogen bonds than water and thus influences g-tensor anisotropy more strongly. Substituent effects at the semiquinone are reproduced quantitatively by the calculations. The g-tensor anisotropy is influenced significantly by the conformations of methyl and methoxy substituents, with opposite contributions. Analyses and interpretations of the interrelations between structure, bonding, and spectroscopic data are provided. The relevance of the computational results for the EPR spectroscopy of semiquinone radical anions in photosynthetic reaction centers is discussed.     

Determining the geometry and magnetic parameters of fluorinated radicals by simulation of powder ESR spectra and DFT calculations: the case of the radical RCF2CF2* in nafion perfluorinated ionomers

The ESR spectrum of the chain-end radical RCF2CF2* detected in Nafion perfluorinated membranes exposed to the photo-Fenton reagent was accurately simulated by an automatic fitting procedure, using as input the hyperfine coupling tensors of the two F alpha and two F beta nuclei as well as the corresponding directions of the principal values from density functional theory (DFT) calculations. An accurate fit was obtained only for different orientations of the hyperfine coupling tensors for the two F alpha nuclei, indicating a nonplanar structure about the C alpha radical center. The fitted isotropic hyperfine splittings for the two F beta nuclei in the Nafion radical, 24.9 and 27.5 G, are significantly larger than those for the chain-end radical in Teflon (15 G), implying different radical conformations in the two systems. The excellent fit indicated that the geometry and electronic structure of free radicals can be obtained not only from single-crystal ESR spectroscopy, but also, in certain cases, from powder spectra, by combination with data from DFT calculations. The optimized structures obtained by DFT calculations for the CF3CF2CF2CF2* or CF3OCF2CF2* radicals as models provided additional support for the pyramidal structure determined from the spectral fit. Comparison and analysis of calculated and fitted values for the hyperfine splittings of the two F beta nuclei suggested that the radical detected by ESR in Nafion is ROCF2CF2*, which originates from attack of oxygen radicals on the Nafion side chain. The combination of spectrum fitting and DFT is considered important in terms of understanding the hyperfine splittings from 19F nuclei and the different conformations of fluorinated chain-end-type radicals RCF2CF2* in different systems, and also for elucidating the mechanism of Nafion fragmentation when exposed to oxygen radicals in fuel cell conditions.     

Q-band EPR and ENDOR of low temperature X-irradiated beta-D-fructose single crystals

Beta-D-fructose single crystals were in situ X-irradiated at 80 K and measured using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques at Q-band (34 GHz) microwave frequencies. The measurements revealed the presence of at least four carbon-centered radicals stable at 80 K. By means of ENDOR angular variations in the three principal crystallographic planes, six proton hyperfine coupling tensors could be determined and were assigned to four different radicals by the aid of EIE. Two of the radicals exhibit only beta-proton hyperfine couplings and reveal almost identical EIE spectra. For the other two radicals, the major hyperfine splitting originates from a single alpha-proton hyperfine coupling and their EIE spectra were also quite similar. The similarity of the EIE spectra and hyperfine tensors led to the assumption that there are only two essentially different radical structures. The radical exhibiting only beta-proton hyperfine couplings was assigned to a C3 centered radical arising from H3 abstraction and the other radical suggested to be an open-ring species with a disrupted C2-C3 bond and a double C2-O2 bond. A possible formation mechanism for the latter open-ring radical is presented. By means of cluster density functional theory (DFT) calculations, the structures of the two radicals were determined and a fairly good agreement between the calculated and experimental hyperfine tensors was found.