Electron Spin Echo Envelope Modulation Spectra Research Articles

Electron Spin-Echo Envelope Modulation Studies on Copper Site of Particulate Methane Monooxygenase fromMethylosinus trichosporiumOB3b

The electron spin-echo envelope modulation (ESEEM) effect was studied on the type II copper site in the particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b. From comparing the ESEEM spectra of the membranes treated with ethylenediaminetetraacetic acid and some model compounds, it was clarified that there were at least two imidazole ligands bound copper (II) in pMMO.

Electron spin echo envelope modulation spectroscopy in mixed alkali silicate glasses

Lithium and sodium silicate glasses of the x mole % Li2O y mole % Na2O (100-x-y) mole % SiO2 composition with x, y between 10–50 were γ-irradiated at room temperature and subsequently studied by electron spin echo envelope modulation (ESEEM) spectroscopy at the X-band and 20 K temperature. Evaluation of the ESEEM spectra revealed weak magnetic coupling between the nonbridging oxygen (NBO) and the neighboring alkali metal. The covalent character between NBO and alkali metal is negligible, though it depends upon the kind weakly. The alkali metal occupies sites forming an angle of 60° with respect to the z-axis of the g-tensor. The magnetic couplings remain unchanged with the variation of the concentration of the alkali or the doping with another alkali.

Simulations of HYSCORE spectra obtained with ideal and non-ideal pulses

The two-dimensional hyperfine-sublevel correlation (HYSCORE) experiment provides correlations between nuclear frequencies belonging to different M S manifolds. It is most useful for the assignment of electron spin echo envelope modulation (ESEEM) frequencies and for the detection of broad signals. A general expression for the echo intensity in the HYSCORE experiment, obtained under the conditions of ideal pulses exists for an S = 1/2 system. In this work it is extended to the case of non-ideal pulses in order to explore the possibility of generating correlations between nuclear frequencies belonging to the same M s manifold, referred to as ‘forbidden’. For this purpose, an effective computer program that calculates the HYSCORE spectrum in the frequency domain under conditions of ideal and non-ideal pulses was developed. The program was used to simulate the HYSCORE spectrum of a frozen solution of a Cu(II) complex with a lipophilic bis-hydroxamate ion binder (Cu-RL252) which exhibits 14N modulations. It is shown that through HYSCORE simulations it is possible to disinguish between two sets of hyperfine parameters which were determined by earlier simulations of a series of orientation-selective ID ESEEM spectra and reproduced the experimental spectra equally well. The experimental HYSCORE spectrum exhibits weak cross-peaks at positions that can be interpreted as correlations within the same M s manifold. The possibility that such ‘forbidden’ HYSCORE correlations are a consequence of non-ideal pulses and off-resonance effects is investigated, and it is shown that such cross-peaks may appear for relatively long pulses but their relative intensities are negligible. Therefore the features observed in the experimental spectra are due to accidental overlapping of nuclear frequencies in the two different M s manifolds. Moreover, the simulations indicate that for hyperfine couplings obeying the cancellation condition at X band and for quadrupolar coupling constants up to ∼4 MHz, pulse durations of t π/2 = t π ≈ 20 ns, which usually are used under experimental conditions, can well be considered as ideal pulses.

Electronic structure ofB2O3glass studied by one- and two-dimensional electron-spin-echo envelope modulation spectroscopy

${\mathrm{B}}_{2}{\mathrm{O}}_{3}$ in glass and crystalline states have been subjected to \ensuremath{\gamma} irradiation at room temperature and subsequently studied by continuous-wave electron paramagnetic resonance and electron-spin-echo envelope modulation (ESEEM) spectroscopy at liquid-helium temperature. The ESEEM study of ${\mathrm{B}}_{2}{\mathrm{O}}_{3}$ revealed the existence of weak magnetic couplings between the irradiation-induced paramagnetic centers and nearby ${}^{10}\mathrm{B},$ ${}^{11}\mathrm{B}$ nuclei. The assignment of the observed couplings was achieved by the use of the two-dimensional hyperfine sublevel correlation spectroscopy. A detailed theoretical analysis of the ESEEM spectra and a $S=\frac{1}{2},$ $I=\frac{3}{2}$ system in both time and frequency domain is also presented. Computer simulation of the spectra, Hartree-Fock self-consistent field, and modified neglect of differential overlap calculations revealed that the unpaired electron in the ${\mathrm{B}}_{2}{\mathrm{O}}_{3}$ glass is associated with a dangling bond of oxygen attached to a boron of a boroxol ring. The paramagnetic centers of the ${\mathrm{B}}_{2}{\mathrm{O}}_{3}$ crystal are associated with oxygen dangling bonds in boron trigonal units. Pertinent structural models for the glass ${\mathrm{B}}_{2}{\mathrm{O}}_{3}$ are examined at a microscopic level.

Double Nuclear Coherence Transfer (DONUT)-HYSCORE: A New Tool for the Assignment of Nuclear Frequencies in Pulsed EPR Experiments

A two-dimensional experiment, termed DONUT-HYSCORE (double nuclear coherence transfer hyperfine sublevel correlation) designed to obtain correlations between nuclear frequencies belonging to the same electron spin manifold is presented. The sequence employed is π/2−τ1−π/2−t1−π−τ2−π−t2−π/2−τ1−echo, and the echo is measured as a function of t1 and t2 whereas τ1 and τ2 are held constant. It is complementary to the standard HYSCORE experiment which generates correlations between nuclear frequencies belonging to different MS manifolds and is particularly useful for 14N nuclei. The experiment is first demonstrated on a single crystal of copper-doped l-histidine hydrochloride monohydrate where the modulations are induced by a single 14N nucleus, the remote nitrogen in the imidazole group. HYSCORE and DONUT-HYSCORE experiments were carried out on two crystal orientations. In the first, one Cu2+ site contributes to the echo and all six nuclear frequencies together with the expected correlation were observed. In the second, 12 frequencies corresponding to two Cu2+ ions at different crystallographic sites appeared and all expected correlations were detected as well. This rather trivial example demonstrates that the DONUT-HYSCORE pulse sequence indeed generates correlations within the MS manifolds. The value of the DONUT-HYSCORE experiment is demonstrated on a frozen solution of a vanadyl complex with a bis-hydroxamate ion binder (VO-RL515). The modulations in this complex arise from the two 14N nuclei in the hydroxamate groups, and orientation-selective three-pulse ESEEM (electron spin−echo envelope modulation) spectra showed a number of well-resolved peaks. An unambiguous assignment of all peaks and their orientation dependences could not be achieved through HYSCORE alone because at certain orientations frequencies of one of the MS manifolds were absent or overlapped with those of the other manifold. The application of the DONUT-HYSCORE experiment provided new correlations that led to the complete assignment of the ESEEM frequencies, thus paving the way for future systematic spectral simulations for the determination of the best-fit Hamiltonian parameters. This example shows that, in the case that the HYSCORE experiment cannot distinguish between two sets of frequencies belonging to the same MS manifold in different centers (or orientations) because signals from the other manifold are missing or overlapping, the DONUT-HYSCORE becomes most valuable.

Electron Spin−Echo Envelope Modulation Spectrum of Azurin at X-Band

Frozen solutions of azurin and of its mutant His117Gly in the presence of [15N]imidazole have been investigated by 9 GHz three-pulse electron spin−echo envelope modulation (ESEEM) and two-dimensional ESEEM spectroscopy. It is found that the interaction of the unpaired electron with a backbone nitrogen leads to a sizable contribution to the ESEEM spectrum. This observation provides the clue to the interpretation of the ESEEM spectrum of the prototypical blue-copper site of azurin. The modulations stem largely from the interaction of the electron spin with three nitrogen nuclei. The quadrupole bands of the remote nitrogen of histidine-117 dominate the spectrum. Smaller but significant contributions result from the backbone nitrogen and the remote nitrogen of histidine-46. Simulations of the ESEEM spectra of azurin confirm this interpretation and yield complete hyperfine and quadrupole tensors for all three nitrogens. The significant contribution of the backbone nitrogen to the ESEEM spectra of azurin nicely brings out the delocalized character of the unpaired-electron wave function of the oxidized copper center.

Electron Spin Echo Envelope Modulation Spectroscopy Reveals and Distinguishes Equatorial and Axial Nitrogen Ligands Bound to VO2+

ESEEM (electron spin echo envelope modulation) spectra of a series of vanadyl complexes have for the first time revealed the spectral features that arise from amine nitrogen donors trans to the vanadyl oxo moiety. The complexes [VO(H2O)ada] (1) and [VO(H2O)Hheida] (2) (H2ada, N-(2-acetamido)iminodiacetic acid; H3heida, N-(2-hydroxyethyl)iminodiacetic acid) have recently been synthesized and crystallographically characterized (Hamstra, B. J.; Houseman, A. L. P.; Colpas, G. J.; Kampf, J. W.; LoBrutto, R.; Frasch, W. D.; Pecoraro, V. L. Inorg. Chem. 1997, 36, 4866−4874). In each structure, the ligand is disposed so that the tertiary nitrogen is bound trans to the vanadyl oxo. In solution, the structural integrity of these compounds is maintained as observed by UV/vis and EPR spectroscopies. ESEEM spectra of 1, 2, and [VO(H2O)nta]- (3) (H3nta, nitrilotriacetic acid) reveal that the superhyperfine coupling constants for axial amines are ≅1.3 MHz as opposed to the ≅5.0 MHz superhyperfine couplings typically observed for equatorially coordinated amines. The data indicate that not only can axial nitrogen donors to VO2+ be detected, but they may also be distinguished from nitrogen donors in the equatorial plane of the vanadyl ion. The implications of this study with respect to understanding the coordination environment of VO2+ in the vanadyl-substituted chloroplast F1-ATPase (CF1) and the reduced, inactive form of vanadium bromoperoxidase (VBrPO) are discussed.

Electron Spin−Echo Envelope Modulation Study of Imidazole-Coordinated Oxovanadium(IV) Complexes Relevant to the Active Site Structure of Reduced Vanadium Haloperoxidases

Electron spin echo envelope modulation (ESEEM) measurements have been performed for VO(Himac)2 (1) and VO(salimH)(acac) (2) (HimacH = 4-imidazoleacetic acid; HsalimH = 4-(2-(salicylideneamino)ethyl)-imidazole). 1 exhibits an ESEEM spectrum quite similar to that of the reduced vanadium bromoperoxidase. The behavior of 2 toward acid has been also investigated. The ESEEM results reveal that the Schiff base nitrogen is detached from vanadium upon addition of 1 equiv of acid.

The distance between P680 and Q A in Photosystem II determined by ESEEM spectroscopy

Light induced spin-polarized P680 +Q − A radical pairs were studied by two pulse electron spin echo envelope modulation (ESEEM) spectroscopy in the cyanide-treated and Zn-substituted Photosystem II core complexes and in the isolated D1-D2-cyt b559 reaction center complexes reconstituted with dibromoisopropyl- p-benzoquinone. The observed strong out-of phase ESEEM signals were identified as those of the P680 +Q − A radical pairs based on the time variation of the transient CW EPR spectra. The shapes of ESEEM spectra were attributed to dipolar D and spin exchange J interactions in the radical pairs. The values of D and J were derived from a sine Fourier transformation and the center-to-center distance between P680 and Q A was determined to be 27.2±1.0 Å for all three preparations.

Hyperfine decoupling in electron spin resonance

A new class of experiments is introduced to electron spin resonance (ESR) spectroscopy that utilizes hyperfine decoupling for resolution enhancement and spectrum simplification, and that provides a basis for correlation techniques. A general framework is provided for the discussion of pulse ESR experiments on systems with arbitrary effective electron spin S and an arbitrary number of coupled nuclear spins and is used to describe spin decoupling in pulse ESR and ENDOR spectroscopy. Analytical expressions are given for the hyperfine-decoupled nuclear frequencies and the residual hyperfine splittings of spin-1/2 nuclei during strong decoupling. Pulse sequences are proposed for hyperfine-decoupled electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) experiments as well as for the correlation of the hyperfine-decoupled ESEEM spectrum with two-pulse and three-pulse ESEEM spectra and of hyperfine-decoupled ENDOR with the hyperfine splittings. It is shown that hyperfine-decoupled ESEEM and ENDOR spectra can reveal information on the magnetic quantum numbers involved in an ESR observer transition, and that choosing a transition mS↔mS+1 with mS≠−1/2 can improve the resolution of a nuclear frequency spectrum. In addition, such experiments can be used to determine the relative signs of hyperfine couplings. The potential of the two-dimensional DECENT (decoupled ESEEM correlated to nuclear transition frequencies) experiment is demonstrated on weakly coupled N14 nuclei in both an ordered and a disordered system and on the hexaquo manganese (II) complex (S=5/2) in a single crystal. It is also shown that for the ESR observer transition mS=(−3/2↔−5/2) the S=5/2 system yields highly resolved hyperfine-decoupled ENDOR spectra which allow for a complete assignment of the ENDOR lines.

One- and Two-Dimensional Electron Spin Echo Envelope Modulation Study of the Intermediate Electron Acceptor Pheophytin in 14N- and 15N-Labeled Photosystem II

The intermediate electron acceptor of the photosystem II (PSII) reaction center, which is a pheophytin-α (Pheo) molecule, was studied by pulsed EPR. Electron spin echo envelope modulation (ESEEM) experiments were carried out in 14N- and 15N-labeled PSII reaction centers. The assignment of the ESEEM frequencies was achieved by the application of three-pulse (stimulated echo) and four-pulse 2D-HYSCORE sequences. Computer simulations of the 14N- and 15N-stimulated ESEEM spectra were performed, yielding a complete set of hyperfine and quadrupole coupling constants of the four nitrogens of the macrocycle of the pheophytin. The nitrogen hyperfine couplings are characterized by considerable anisotropy while the isotropic part is either smaller or much larger than the nuclear Larmor frequency. The present results show that the spin density distribution on the Pheo- in PSII is highly asymmetric. In agreement with earlier theoretical predictions for Pheo- in vitro, the ESEEM data show that in PSII the two unprotonated nitrogens bear considerable spin density while the protonated nitrogens bear a small fraction of the spin density.

Probing the energy landscape of bacterial photosynthetic reaction centers at cryogenic temperatures by ESEEM of spin-polarised D +Q A− radical pairs

The energy landscape of the reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides R-26 has been investigated with electron spin echo envelope modulation (ESEEM) spectroscopy of the modulations induced by spin-spin interactions (dipolar and exchange) between radicals in the light-induced spin-polarized radical pair D +Q A − (D, primary electron donor; Q A, primary electron acceptor). At temperatures above ∼ 100 K the values of the dipolar and spin-exchange coupling between D + and Q A − were found to be −(115 ± 5) μT and 0.7 μT (with uncertainty of +1.5 and −0.3 μT), respectively. Abrupt changes of the linewidth of the Fourier-transformed ESEEM spectrum were observed near 25, 40 and 80 K. The lineshapes could be simulated assuming that the distance between the two radicals is distributed within a range of about 4 Å and that the distribution depends stepwise on the temperature. The similarity between our results and those obtained with optical spectroscopies on Zn-protoporphyrin substituted myoglobin suggests that the stepwise changes in distance distribution are related to relaxation along a hierarchical self-similar pattern of minima in the multidimensional potential surface of the protein, and that this energy landscape is a global property of the protein.

Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum

The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA. Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14N nuclei magnetically coupled to the paramagnet. In 2H 2O buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre. Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system. The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.

FTIR and EPR characterisation of copper-exchanged mordenites and beta zeolites

Sodium mordenites and beta zeolites, partially exchanged with Cu 2+ , were characterised by means of Fourier transform infrared (FTIR) and both continuous-wave and pulse electron paramagnetic resonance (EPR) spectroscopies, in order to shed light on the catalytic role of copper ions in the ammoxidation of 1-methylnaphthalene. FTIR analysis showed that ammonia interacts with both Bronsted and Lewis acid sites, while 1-methylnaphthalene is mainly coordinated through π-complexes. The acidity of the catalysts was also determined through FTIR analysis of the adsorbed pyridine. The main changes in the coordination sphere of Cu 2+ ions were observed by EPR analysis after ammonia adsorption and after catalyst use, while adsorption of 1-methylnaphthalene had a very minor effect. Primary electron spin echo envelope modulation spectra after catalytic use showed the presence of organic radicals, having a relaxation time of longer than the Cu 2+ ions. The latter are confirmed as the active reaction centres and should be located in the side pockets of the mordenites, and be unaccessible to the aromatic system of 1-methylnaphthalene. This explains the higher selectivity observed with mordenites. In the more acidic beta zeolites the presence of copper ions on the walls of large channels initially favours side reactions, but also makes adsorbed ammonia more available for ammoxidation.

Evidence that Ala M260 is hydrogen-bonded to the reduced primary acceptor quinone Q A−. in reaction centers of Rb. sphaeroides

The binding of the primary quinone anion radical Q A −. in reaction centers (Rcs) of Rhodobacter (Rb.) sphaeroides species was investigated with electron spin echo envelope modulation (ESEEM). The ESEEM spectra, at two microwave frequencies, of Zn-substituted RCs of Rb. sphaeroides R26 showed interactions of the unpaired electron of Q A −. with two nitrogen nuclei in the protein matrix. From an analysis of the experimental data the nitrogen nuclear quadrupole resonance parameters were determined: e 2 qQ/ h = 1.52 MHz, η = 0.82 and e 2 qQ/ h = 3.04 MHz, η = 0.66, which are assigned to the 14N δ(1)N group of His M219 and the peptide 14N of Ala M260. The ESEEM spectrum of Q A −. in reaction centers of the Rb. sphaeroides mutant W(M252)Y shows that the nitrogen of Trp M252 is not interacting with Q A −., that of the mutant H(M266)C shows that manipulating the Fe binding site strongly affects the Q A-binding site.

Zero-field electron spin echo envelope modulation spectroscopy

Two-pulse and three-pulse electron spin echo envelope modulation (ESEEM) patterns and pulse ENDOR spectra have been observed at zero magnetic field. It is shown theoretically that for systems with half-integer electron spin quantum numbers optimum conditions for zero-field ESEEM are expected for the observer frequency at an electron spin transition between states with magnetic quantum numbers m s = ± 1 2 and m s = ± 3 2 . Furthermore, it is found that a compensation of the earth field is not needed for the measurement of unperturbed nuclear frequencies in the m s = ± 3 2 manifolds. From the three-pulse zero-field ESEEM spectrum of the [MnCl 4(H 2O) 2] 2− center in polycrystalline ammonium chloride, hyperfine and nuclear quadrupole couplings of the four nearest neighbour chlorines of the manganese ion have been evaluated.

Identification of a Silver Hydroxymethyl Radical in Ag-NaA Zeolite Using Electron Spin Echo Envelope Modulation Spectroscopy

In Ag-loaded NaA zeolite the silver hydroxymethyl radical (Ag•CH2OD)+ was stabilized and studied with electron spin echo envelope modulation (ESEEM) spectroscopy. Careful control of the CH3OD concentration enabled the measurement of ESEEM spectra from which the hyperfine and quadrupole parameters of the intramolecular hydroxyl deuterium nucleus could be estimated. The isotropic hyperfine splitting constant, Aiso, is equal to +2.9 MHz, the electron−deuteron dipolar coupling parameter T (T = geβegDβD/(hr3)) is equal to +1.4 MHz, and the quadrupole parameter P (P = e2qQ/(4h)) is equal to ±0.12 MHz. These data are compared to listed data for the •CH2OH radical. The differences can be interpreted in terms of electronic, electrostatic, and matrix effects.

Magnetic Field (g-Value) Dependence of Proton Hyperfine Couplings Obtained from ESEEM Measurements: Determination of the Orientation of the Magnetic Axes of Model Heme Complexes in Glassy Media

Electron spin echo envelope modulation (ESEEM) studies were utilized to characterize the coupling between protons of axially bound pyrazole ligands (PzH) and the unpaired electron of low-spin tetraphenylporphyrinatoiron(III) chloride. Samples were prepared in mixed-solvent glasses to maximize the resolution of the electron paramagnetic resonance (EPR) signals. X-band two-pulse ESEEM experiments at 4.2 K in deuterated solvent glasses demonstrated that this coupling results in 0.2−0.7 MHz shifts of the να + νβ proton sum combination peak from twice the Larmor frequency. These shifts have been investigated across most of the EPR absorption spectrum of [TPPFe(PzH)2]+Cl-. Two-pulse ESEEM spectra were simulated at different magnetic field positions. Combination peaks were observed from both distant protons (DP) (β-pyrrole, ortho-phenyl, and β-pyrazole) and near protons (NP) (α-H of the pyrazole ligands). For the simulations, the orientation of the nearest four protons of the pyrazole ligands with respect to the...

A new one-dimensional correlation spectroscopy (1D COSY) for electron spin echo envelope modulation studies

A new one-dimensional correlation spectroscopy (COSY) method for electron spin echo envelope modulation (ESEEM) studies is developed based on introducing a general signal-acquisition scheme to detect both sum-frequency and difference-frequency 1D ESEEM spectra. Using a single crystal of Cu/Ni(bu 2dtc) 2 at 21 K, it is shown that the correlated nuclear transitions can be easily determined. To compare with existing 2D ESEEM schemes, it is experimentally demonstrated that higher sensitivity and resolution can be easily achieved by using this new 1D ESEEM COSY method. A graphical approach to using this spectroscopic method is also described.

Coordination of Cu(II) to Lipophilic Bis-hydroxamate Binders As Studied by One- and Two-Dimensional Electron Spin Echo Spectroscopy

The binding of Cu(II) to a lipophilic bis-hydroxamate binder, RL252, and its parent RL239, was investigated by pulsed EPR. The binders have two arms, each terminated with a hydroxamate group which serves as a donor. The major difference between RL252 an RL239 is the absence of the leucine amino acid bridge in RL239. Orientation-selective electron spin echo envelope modulation (ESEEM) experiments were carried out at 8.45 and 9.15 GHz. The spectra obtained were exceptionally well resolved and indicated that the cancellation condition, which requires that the hyperfine coupling is approximately twice the nuclear Larmor frequency, is met at 8-9 GHz. The spectra of both Cu-RL252 and Cu-RL239 showed the nuclear quadrupole resonance (NQR) frequencies, v{sub o}, v{sub -}, and v{sub +}, of the {sup 14}N of the hydroxamate groups at 1.6-1.7, 2.15-2.25, and 3.8-3.9 MHz. A peak corresponding to the double quantum transition in the `noncanceled manifold` v{sub DQ}, was observed at 5-5.2 MHz, and at some magnetic fields a peak corresponding to a single quantum transition, v{sub SQ}, and its combination harmonic appeared as well. The assignment of all the ESEEM frequencies was achieved by the application of the two-dimensional hyperfine sublevel correlation (HYSCORE) experiment. Following the assignment, simulationsmore » of the orientation-selective ESEEM spectra were performed, yielding the magnitude and orientation of the quadrupole and hyperfine tensors of the coupled nitrogens. 52 refs., 9 figs., 3 tabs.« less