Electron Nuclear Double Resonance Data Research Articles

Pulsed Electron-Nuclear Double Resonance in the Fourier Regime.

Nuclear magnetic resonance (NMR) spectroscopy provides atomic-level molecular structural information. However, in molecules containing unpaired electron spins, NMR signals are difficult to measure directly. In such cases, data is obtained using the electron-nuclear double resonance (ENDOR) method, where nuclei are detected through their interaction with nearby unpaired electron spins. Unfortunately, electron spins spread the ENDOR signals, which challenges current acquisition techniques, often resulting in low spectral resolution that provides limited structural details. Here, we show that by using miniature microwave resonators to detect a small number of electron spins, integrated with miniature NMR coils, one can excite and detect a wide bandwidth of ENDOR data in a single pulse. This facilitates the measurement of ENDOR spectra with narrow lines spread over a large frequency range at much better spectral resolution than conventional approaches, which helps reveal details of the paramagnetic molecules' chemical structure that were not accessible before.

Statistical analysis of ENDOR spectra

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Structures of Impurity Defects in Lithium Niobate and Tantalate Derived from Electron Paramagnetic and Electron Nuclear Double Resonance Data

Point intrinsic and extrinsic defects, especially paramagnetic ions of transition metals and rare-earth elements, have essential influence on properties of lithium niobate, LN and tantalate, LT, and often determine their suitability for numerous applications. Discussions about structures of the defects in LN/LT have lasted for decades. Many experimental methods facilitate progress in determining the structures of impurity centers. This paper gives current bird’s eye view on contributions of Electron Paramagnetic Resonance (EPR), and Electron Nuclear Double Resonance (ENDOR) studies to the determination of impurity defect structures in LN and LT crystals for a broad audience of researchers and students. Symmetry and charge compensation considerations restrict a number of possible structures. Comparison of measured angular dependences of ENDOR frequencies with calculated ones for Li and Nb substitution using dipole–dipole approximation allows unambiguously to determine the exact location of paramagnetic impurities. Models with two lithium vacancies explain angular dependencies of EPR spectra for Me3+ ions substituting for Li+ like Cr, Er, Fe, Gd, Nd, and Yb. Self-compensation of excessive charges through equalization of concentrations of Me3+(Li+) and Me3+(Nb5+) and appearance of interstitial Li+ in the structural vacancy near Me3+(Nb5+) take place in stoichiometric LN/LT due to lack of intrinsic defects.

Influence of Al on the Structure and in Vitro Behavior of Hydroxyapatite Nanopowders.

Nanopowders of aluminum-substituted (0-20 mol %) hydroxyapatite (HA) with the average size of 40-60 nm were synthesized by the precipitation method from nitrate solutions. A series of samples were studied by various analytical tools to elucidate the peculiarities of Al introduction. Electron paramagnetic resonance and pulsed electron-nuclear double resonance data demonstrate that incorporation of Al resulted in a decrease in the concentration of impurity carbonate anions and lead to an increase in the number of protons in the distant environment of the impurity nitrogen species. Density functional theory calculations show that the Al3+ incorporation is accompanied by the local positional rearrangement and the distortion of anion channel geometry. An in vitro test conducted on MG-63 cells demonstrates the cytocompatibility and magnification of the surface matrix characteristics with Al doping.

Reconciling Structural and Spectroscopic Fingerprints of the Oxygen-Evolving Complex of Photosystem II: A Computational Study of the S2 State.

The catalytic cycle of photosynthetic water oxidation occurs at the Mn4CaO5 oxygen-evolving complex (OEC) of photosystem II. Extensive spectroscopic data have been collected on the intermediates, especially the S2 (Kok) state, although the proton and electron inventories (Mn oxidation states) are still uncertain. The "high oxidation" paradigm assigns S2 Mn oxidation level (III, IV, IV, IV) or (IV, IV, IV, III), whereas a "low oxidation" paradigm posits two additional electrons. Here, we investigate the geometric (X-ray diffraction, extended X-ray absorption fine structure) and spectroscopic (electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR)) properties of the S2 state using quantum chemical density functional theory calculations, focusing on the neglected low paradigm. Two interconvertible electronic spin configurations are predicted as ground states, producing multiline ( S = 1/2) and broad ( S = 5/2) EPR signals in the low paradigm oxidation state (III, IV, III, III) and with W2 as OH- and O5 as OH-. They have "open" ( S = 5/2) and "closed" ( S = 1/2) Mn3CaO4-cubane geometries. Other energetically accessible isomers with ground spin states 1/2, 7/2, 9/2, or 11/2 can be obtained through perturbations of hydrogen-bonding networks (e.g., H+ from His337 to O3 or W2), consistent with experimental observations. Conformers with the low oxidation state configuration (III, IV, IV, II) also become energetically accessible when the protonation states are O5 (OH-), W2 (H2O), and neutral His337. The configuration with (III, IV, III, III) agrees well with earlier low-temperature EPR and ENDOR interpretations, whereas the MnII-containing configuration agrees partially with recent ENDOR data. However, the low oxidation paradigm does not yield isotropic ligand hyperfine interactions in good agreement with observed values. We conclude that the low Mn oxidation state proposal for the OEC can closely fit most of the available structural and electronic data for S2 at accessible energies.

Composition and Structure of the Inorganic Core of Relaxed Intermediate X(Y122F) of Escherichia coli Ribonucleotide Reductase.

Activation of the diferrous center of the β2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied (57)Fe, (14,15)N, (17)O, and (1)H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of (17)O and (1)H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH(-))Fe(III)-O-Fe(IV)]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the (14)N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238.

Insertion of lithium ions into TiO2 (rutile) crystals: An electron paramagnetic resonance study of the Li-associated Ti3+ small polaron

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) are used to identify a Ti3+-Li+ complex in TiO2 crystals having the rutile structure. This defect consists of an interstitial Li+ ion adjacent to a substitutional Ti3+ ion (the unpaired electron on the Ti3+ ion provides charge compensation for the Li+ ion). The neutral Ti3+-Li+ complex is best described as a donor-bound small polaron and is similar in structure to the recently reported neutral fluorine and hydrogen donors in TiO2 (rutile). Lithium ions are diffused into the crystals at temperatures near 450 °C. Following the diffusion, an EPR spectrum containing groups of four closely spaced lines is observed at 36 K without laser illumination. ENDOR data verify that the four lines within each group are due to a weak hyperfine interaction with one lithium nucleus. Spin-Hamiltonian parameters are obtained from the angular dependence of the EPR spectra. Principal values are 1.9688, 1.9204, and 1.9323 for the g matrix and –2.14, –2.20, and +3.44 MHz for the 7Li hyperfine matrix.

Detection of the Water-Binding Sites of the Oxygen-Evolving Complex of Photosystem II Using W-Band17O Electron–Electron Double Resonance-Detected NMR Spectroscopy

Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).

Measuring central-spin interaction with a spin bath by pulsed ENDOR: Towards suppression of spin diffusion decoherence

We present pulsed electron-nuclear double resonance (ENDOR) experiments which enable us to characterize the coupling between bismuth donor spin-qubits in Si and the surrounding spin-bath of (29)Si impurities which provides the dominant decoherence mechanism (nuclear spin diffusion) at low temperatures (< 16 K). Decoupling from the spin-bath is predicted and cluster correlation expansion simulations show near-complete suppression of spin diffusion, at optimal working points. The suppression takes the form of sharply peaked divergences of the spin diffusion coherence time, in contrast with previously identified broader regions of insensitivity to classical fluctuations. ENDOR data shows anisotropic contributions are comparatively weak, so the form of the divergences is independent of crystal orientation.

Synthetic model of the asymmetric [Mn 3 CaO 4 ] cubane core of the oxygen-evolving complex of photosystem II

The laboratory synthesis of the oxygen-evolving complex (OEC) of photosystem II has been the objective of synthetic chemists since the early 1970s. However, the absence of structural information on the OEC has hampered these efforts. Crystallographic reports on photosystem II that have been appearing at ever-improving resolution over the past ten years have finally provided invaluable structural information on the OEC and show that it comprises a [Mn(3)CaO(4)] distorted cubane, to which is attached a fourth, external Mn atom, and the whole unit attached to polypeptides primarily by aspartate and glutamate carboxylate groups. Such a heterometallic Mn/Ca cubane with an additional metal attached to it has been unknown in the literature. This paper reports the laboratory synthesis of such an asymmetric cubane-containing compound with a bound external metal atom, [(1)]. All peripheral ligands are carboxylate or carboxylic acid groups. Variable-temperature magnetic susceptibility data have established 1 to possess an S = 9/2 ground state. EPR spectroscopy confirms this, and the Davies electron nuclear double resonance data reveal similar hyperfine couplings to those of other Mn(IV) species, including the OEC S(2) state. Comparison of the X-ray absorption data with those for the OEC reveal 1 to possess structural parameters that make it a close structural model of the asymmetric-cubane OEC unit. This geometric and electronic structural correspondence opens up a new front in the multidisciplinary study of the properties and function of this important biological unit.

Oxygen vacancies adjacent to Cu2+ ions in TiO2 (rutile) crystals

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) are used to characterize Cu2+ ions substituting for Ti4+ ions in nominally undoped TiO2 crystals having the rutile structure. Illumination at 25 K with 442 nm laser light reduces the concentration of Cu2+ ions by more than a factor of 2. The laser light also reduces the EPR signals from Fe3+ and Cr3+ ions and introduces signals from Ti3+ ions. Warming in the dark to room temperature restores the crystal to its preilluminated state. Monitoring the recovery of the photoinduced changes in the Cu2+ ions and the other paramagnetic electron and hole traps as the temperature is raised from 25 K to room temperature provides evidence that the Cu2+ ions have an adjacent doubly ionized oxygen vacancy. These oxygen vacancies serve as charge compensators for the substitutional Cu2+ ions and lead to the formation of electrically neutral Cu2+-VO complexes during growth of the crystals. The Cu2+-VO complexes act as electron traps and convert to nonparamagnetic Cu+-VO complexes when the crystals are illuminated at low temperature. Complete sets of spin-Hamiltonian parameters describing the electron Zeeman, hyperfine, and nuclear electric quadrupole interactions for both the 63Cu and 65Cu nuclei are obtained from the EPR and ENDOR data. This study suggests that other divalent cation impurities in TiO2 such as Co2+ and Ni2+ may also have an adjacent oxygen vacancy for charge compensation.

Revisiting the nitrosyl complex of myoglobin by high-field pulse EPR spectroscopy and quantum mechanical calculations

The binding of NO to reduced myoglobin in solution results in the formation of two paramagnetic nitrosyl myoglobin (MbNO) complexes: one with a rhombic g-factor and the other with an axial one, referred to as the R- and A-forms. In spite of past extensive studies of MbNO by crystallography, spectroscopy and quantum chemical calculations it is still not clear what factors determine the appearance of the two forms. In this work we applied a combination of state of the art quantum chemical calculations and high field pulsed EPR spectroscopy (W-band, 3.4 T/95 GHz) to further characterize the two forms. Specifically, we have used (1)H and (2)H electron-nuclear double resonance (ENDOR) spectroscopy to identify and characterize the H-bond to the NO, and hyperfine sub-level correlation (HYSCORE) spectroscopy to determine the hyperfine and quadrupole interactions of the Fe(ii) coordinated (14)N of the proximal histidine (14)N(His93). The calculations employed quantum mechanics (QM), particularly density functional theory (DFT) methods in combination with molecular mechanics (MM) force-field to model the protein environment. Through QM/MM calculations of the EPR parameters we have explored their dependence on several geometrical factors of the Fe-NO bond and found those that reproduce the best experimental results. The spread of the W-band EPR spectrum of MbNO due to the g-anisotropy is large and there is a significant part of the spectrum where the R-form is the sole contributor. This allowed us to resolve some new characteristics of the R-form: (i) a NO-H hydrogen bond has been detected and characterized and through QM/MM calculations has been unambiguously assigned to (epsilon2)H(His64). (ii) The complete hyperfine and quadrupole interactions of (14)N(His93) have been determined and correlated with structural parameters again using QM/MM calculations. The agreement between the experimental results and calculations varied between excellent and good, depending on the EPR parameter in question. As for the more elusive A-form, the results only suggest that it does have a (14)N(His93) ligand with a hyperfine comparable to that of the R-form and it has less hydrogen bonding interaction with His(64). The calculations also established the orientation of the principal g-values, finding that they are closely related to the orientation of the NO bond. This information is essential for deriving structural information from the experimental orientation selective HYSCORE and ENDOR data.

The Electronic State of Flavoproteins: Investigations with Proton Electron–Nuclear Double Resonance

Electron-nuclear double resonance (ENDOR) spectroscopy provides useful information on hyperfine interactions between nuclear magnetic moments and the magnetic moment of an unpaired electron spin. Because the hyperfine coupling constant reacts quite sensitively to polarity changes in the direct vicinity of the nucleus under consideration, ENDOR spectroscopy can be favorably used for the detection of subtle protein-cofactor interactions. A number of pulsed ENDOR studies on flavoproteins have been published during the past few years; most of them were designed to characterize the flavin cofactor by means of its protonation state, or to detect individual protein-cofactor interactions. The aim of this study is to compare the pulsed ENDOR spectra from different flavoproteins in terms of variations of characteristic proton hyperfine values. The general concept is to observe limits of possible influences on the cofactor's electronic state by surrounding amino acids. Furthermore, we compare ENDOR data obtained from in vivo experiments with in vitro data to emphasize the potential of the method for gaining molecular information in complex media.

Electronic structure of the tyrosine D radical and the water-splitting complex from pulsed ENDOR spectroscopy on photosystem II single crystals

Pulsed electron nuclear double resonance (ENDOR) spectroscopy at Q- and W-band frequencies was applied to single crystals of photosystem II from Th. elongatus. W-Band (1)H-ENDOR on the dark-stable radical state Y of the redox-active tyrosine residue Y(D) yields a complete mapping of the electronic structure of this amino acid radical in terms of an assignment of all hyperfine coupling tensors of the protons covalently bound to the side chain. This study can serve as a model case for the potential of high-field/high-frequency ENDOR on protein single crystals for obtaining highly resolved electronic structure information. Q-band (55)Mn-ENDOR was applied to the S(2) oxidation state of the water-splitting complex in photosystem II single crystals. Irrespective of the difficulties arising from the extremely broad electron paramagnetic resonance (EPR) spectroscopy ( approximately 200 mT) and ENDOR ( approximately 100 MHz) spectra a tentative assignment of the Mn ion in the formal oxidation state III to a Mn position in the structural model of PSII is possible on the basis of the ENDOR data.

Electron paramagnetic resonance and electron‐nuclear double resonance of nonequivalent Yb3+ centers in stoichiometric lithium niobate

AbstractLithium niobate crystals doped with ytterbium were studied using Electron Paramagnetic Resonance (EPR) and Electron Nuclear Double Resonance (ENDOR). The tremendous narrowing of EPR lines in nearly stoichiometric samples, when compared to those in congruent samples, allowed us to distinguish nine non‐equivalent centers, as well as line splitting caused by the hyperfine interaction of ytterbium electrons with the nuclear spins of two magnetic isotopes, 171Yb and 173Yb. Eight of the nine centers are described for the first time. It was found that three of the centers have axial C3 symmetry, and all others have the lowest C1 symmetry due to the presence of intrinsic defects and/or charge compensation defects in the near neighborhood of Yb3+. Characteristics of the g ‐tensor for all of the centers and hyperfine tensors for axial centers were determined. The ENDOR observations of Nb nuclei in the nearest neighborhood of Yb13+ gave direct evidence that the dominated axial Yb1 center has no charge compensator in its nearest surroundings (distant charge compensation mechanism). Both the EPR and ENDOR data for the main axial ytterbium center are explained by a supposition that Yb3+ ions substitute for Li+. Possible models for low‐symmetry centers are discussed. The obtained numerous spectroscopic parameters can be used as cornerstones for model calculations of Yb3+ centers in lithium niobate. (© 2009 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Integrated Paramagnetic Resonance of High-Spin Co(II) in Axial Symmetry: Chemical Separation of Dipolar and Contact Electron−Nuclear Couplings

Integrated paramagnetic resonance, utilizing electron paramagnetic resonance (EPR), NMR, and electron-nuclear double resonance (ENDOR), of a series of cobalt bis-trispyrazolylborates, Co(Tp ( x )) 2, are reported. Systematic substitutions at the ring carbons and on the apical boron provide a unique opportunity to separate through-bond and through-space contributions to the NMR hyperfine shifts for the parent, unsubstituted Tp complex. A simple relationship between the chemical shift difference (delta H - delta Me) and the contact shift of the proton in that position is developed. This approach allows independent extraction of the isotropic hyperfine coupling, A iso, for each proton in the molecule. The Co..H contact coupling energies derived from the NMR, together with the known metrics of the compounds, were used to predict the ENDOR couplings at g perpendicular. Proton ENDOR data is presented that shows good agreement with the NMR-derived model. ENDOR signals from all other magnetic nuclei in the complex ( (14)N, coordinating and noncoordinating, (11)B and (13)C) are also reported.

Pulsed EPR and DFT Characterization of Radicals Produced by Photo-Oxidation of Zeaxanthin and Violaxanthin on Silica−Alumina

Pulsed electron nuclear double resonance (ENDOR) and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) studies in combination with density functional theory (DFT) calculations revealed that photo-oxidation of natural zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor) on silica-alumina produces the carotenoid radical cations (Car*+) and also the neutral carotenoid radicals (#Car*) as a result of proton loss (indicated by #) from the C4(4') methylene position or one of the methyl groups at position C5(5'), C9(9'), or C13(13'), except for violaxanthin where the epoxide at positions C5(5')-C6(6') raises the energy barrier for proton loss, and the neutral radicals #Car*(4) and #Car*(5) are not observed. DFT calculations predict the largest isotropic beta-methyl proton hyperfine couplings to be 8 to 10 MHz for Car*+, in agreement with previously reported hyperfine couplings for carotenoid pi-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic alpha-proton hyperfine coupling tensors determined from the HYSCORE analysis were assigned on the basis of DFT calculations with the B3LYP exchange-correlation functional and found to arise not only from the carotenoid radical cation but also from carotenoid neutral radicals, in agreement with the analysis of the pulsed ENDOR data. The formation of the neutral radical of zeaxanthin should provide another effective nonphotochemical quencher of the excited state of chlorophyll for photoprotection in the presence of excess light.

Characterization of radical intermediates in laccase-mediator systems. A multifrequency EPR, ENDOR and DFT/PCM investigation

Suitable low molecular-weight compounds, called mediators, can be used in combination with the phenol-oxidase enzyme laccase to indirectly oxidize large organic substrates, such as environmental pollutants, which are not laccase natural substrates. The oxidation of two different synthetic redox mediators, violuric acid (VIO) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) has been studied under catalysis of two laccases from white-rot fungi (Trametes versicolor and Pleurotus ostreatus). VIO was selected as a prototype of the -NOH type of mediators and compared to ABTS, a well-known two-step redox system. To characterize the radical intermediates formed from both mediators after the enzymatic oxidation, a multifrequency EPR approach has been adopted. The radical species have been investigated employing 9.4 GHz (X-band), 34 GHz (Q-band) and 244 GHz (high field) EPR and pulse electron nuclear double resonance (ENDOR) techniques. Theoretical calculations based on density functional theory (DFT/PCM) have been performed to support and further interpret the experimental EPR and ENDOR data. This integrated approach allowed us to obtain a complete characterization of both radicals and to elucidate the type of the radical state (neutral or cationic).

Single Crystal 55Mn ENDOR of Concanavalin A: Detection of Two Mn2+ Sites with Different 55Mn Quadrupole Tensors

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. 55Mn electron-nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, ~3.5 T) to determine the 55Mn nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the 55Mn ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e2Qq/h, are significantly different; 10.7 +/- 0.6 MHz for Mand only -2.7 +/-0.6 MHz for M. The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for M and -263.5 MHz for M. The principal z-axis for M is not aligned with any of the Mn-ligand directions, but is 25 off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of M the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90 with respect to MnA. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e2Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally.

Radical-Induced Generation of Small Silver Particles in SPEEK/PVA Polymer Films and Solutions: UV−Vis, EPR, and FT-IR Studies

The present study is centered on the processes involved in the photochemical generation of nanometer-sized Ag particles via illumination at 350 nm of aqueous solutions and cross linked films containing sulfonated poly(ether ether ketone) and poly(vinyl alcohol). Optical and electron paramagnetic resonance experiments, including electron nuclear double resonance data, proved conclusively that the photogenerated chromophore exhibiting a band with lambda(max) = 565 nm is an alpha-hydroxy aromatic (ketyl) radical of the polymeric ketone. This reducing species was produced by illumination of either solutions or films, but the radical lifetime extended from minutes in the fluid phase to hours in the solid. Direct evidence is presented that this long-lived chromophore reduces Ag(I), Cu(II), and Au(III) ions in solution. A rate constant of k = 1.4 x 10(3) M(-)(1) s(-)(1) was obtained for the reduction of Ag(+) by the ketyl radical from the post-irradiation formation of Ag crystallites. FTIR results confirmed that the photoprocess yielding polymeric ketyl radicals involves a reaction between the macromolecules. The photochemical oxidation of the polymeric alcohol, as well as the formation of light-absorbing macromolecular products and polyols, indicates that the sulfonated polyketone experienced transformations similar to those encountered during illumination of the benzophenone/2-propanol system.