Electron nuclear quadruple resonance for assignment of overlapping spectra

Multiple resonance methods are important tools in EPR for revealing the network of hyperfine levels of free radicals and paramagnetic centers. The variations of electron nuclear double resonance (ENDOR) or electron spin-echo envelope modulation (ESEEM) techniques help to correlate nuclear frequencies with each other. These methods have limited utility when there is extensive overlap or suspected overlap in the EPR spectrum between different species or different orientations. In the ENDOR spectrum, overlap and second-order shifts of lines also leads to ambiguity in assignment and interpretation. A new electron nuclear multiple resonance method is presented here that is based on population transfer ENDOR. It is a quadruple resonance method that correlates ENDOR lines and reveals the network of hyperfine levels in samples with unoriented paramagnetic species and in samples with overlapping EPR or ENDOR lines.     

Two-dimensional pulsed TRIPLE at 95 GHz

The one-dimensional (1D) pulsed TRIPLE resonance experiment, introduced by Mehring et al. (M. Mehring, P. Hofer, and A. Grupp, Ber. Bunseges. Phys. Chem. 91, 1132-1137 (1987)) is a modification of the standard Davies ENDOR experiment where an additional RF pi-pulse is applied during the mixing time. While the first RF pulse is set to one of the ENDOR transitions, the frequency of the second RF pulse is scanned to generate the TRIPLE spectrum. The difference between this spectrum and the ENDOR spectrum yields the difference TRIPLE spectrum, which exhibits only ENDOR lines that belong to the same M(S) manifold as the one selected by the first RF pulse. We have extended this experiment in two dimensions (2D) by sweeping the frequencies of both RF pulses. This experiment is particularly useful when the spectrum is congested and consists of signals originating from different paramagnetic centers. The connectivities between the peaks in the 2D spectrum enable a straightforward assignment of the signals to their respective centers and M(S) manifolds, thus providing the relative signs of hyperfine couplings. Carrying out the experiment at high fields has the additional advantage that nuclei with different nuclear gyromagnetic ratios are well separated. This is particularly true for protons which appear at significantly higher frequencies than other nuclei. The feasibility and effectiveness of the experiment is demonstrated at W-band (94.9 GHz) on a crystal of Cu(2+)-doped l-histidine. Homonuclear (1)H-(1)H, (14)N/(35)Cl-(14)N/(35)Cl and heteronuclear (1)H-(14)N/(35)Cl 2D TRIPLE spectra were measured and from the various connectivities in the 2D map the (1)H, (14)N, and (35)Cl signals that belong to two different Cu(2+) centers were identified and grouped according to their M(S) manifolds. Copyright 2000 Academic Press.     

2D TRIPLE in orientationally disordered samples--a means to resolve and determine relative orientation of hyperfine tensors

The two-dimensional (2D) TRIPLE experiment provides correlations between electron-nuclear double resonance (ENDOR) frequencies that belong to the same electron-spin manifold, M(S), and therefore allows to assign ENDOR lines to their specific paramagnetic centers and M(S) manifolds. This, in turn, also provides the relative signs of the hyperfine couplings. So far this experiment has been applied only to single crystals, where the cross-peaks in the 2D spectrum are well resolved with regular shapes. Here we introduce the application of the 2D TRIPLE experiment to orientationally disordered systems, where it can resolve overlapping powder patterns. Moreover, analysis of the shape of the cross-peaks shows that it is highly dependent on the relative orientation of the hyperfine tensors of the two nuclei contributing to this particular peak. This is done initially through a series of simulations and then demonstrated experimentally at a high field (W-band, 95 GHz). The first example concerned the (1)H hyperfine tensors of the stable radical alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) immobilized in a polystyrene matrix. Then, the experiment was applied to a more complex system, a frozen solution of Cu(II)-bis(2,2':6',2' terpyridine) complex. There, the 2D TRIPLE experiment was combined with the variable mixing time (VMT) ENDOR experiment, which determined the absolute sign of the hyperfine couplings involved, and orientation selective ENDOR experiments. Analysis of the three experiments gave the hyperfine tensors of a few coupled protons.     

ESR, ENDOR, and ESEEM investigations on UV-irradiated 2,4,6-tri-tert-butyl phenol crystals

Electron spin resonance (ESR), electron nuclear double resonance (ENDOR), and electron spin echo envelope modulation (ESEEM) measurements were carried out for UV-irradiated 2,4,6-tri-tert-butyl phenol in the polycrystalline state. The radical produced in the crystal was detected by ESR and identified to be the corresponding phenoxyl radical, which is well characterized in the chemical oxidations in solutions. ENDOR and ESEEM spectra were unambiguously analyzed in terms of the hyperfine coupling constants determined from well-resolved ESR in solutions. Radical pairs in the crystals were also ascertained, and together with the single-crystal study the analysis disclosed zero-field splitting parameters in the triplet states. ESEEM time decays gave relaxation timesT ₁ = 5.94 andT ₂ = 1.12 μs at room temperature. These appropriate values permit an easy detection of the spin echoes, and therefore this radical matrix can be used as a useful standard for pulsed ESR investigations.     

Hyperfine decoupling in electron paramagnetic resonance as a powerful tool for unraveling complicated ESEEM spectra of S=1/2, I> or =1/2 systems

Hyperfine decoupling in electron paramagnetic resonance after strong microwave radiation is studied for S=1/2, I=1/2 and S=1/2, I=1 spin systems. A new 2D pulse sequence based on the hyperfine-decoupled DEFENCE (deadtime-free ESEEM by nuclear coherence-transfer echoes) experiment is introduced, which is distinguished by a remarkable reduction of the residual hyperfine coupling. The efficiency of this new decoupling experiment in comparison to the old pulse sequence is studied by means of numerical simulations. The advantages of the new decoupling experiment and its ability to simplify ESEEM spectra are experimentally demonstrated on two disordered systems.     

Low-frequency NMR with a non-resonant circuit

General expressions describing the refocused stimulated (RS) and refocused virtual (RV) electron spin echo envelope modulations (ESEEM) generated with the same basic four-pulse sequence are derived. It is shown that integration of the 3D time domain trace over the two "low-resolution" time intervals (those between the first and second and between the third and fourth microwave pulses) results in a dead time-free 1D ESEEM trace in the "high-resolution" dimension (i.e., the time interval between the second and third microwave pulses) that only contains harmonics with the fundamental frequencies of nuclear transitions. The practical implementation of the integrated RS ESEEM requires pulse swapping, which leads to unrecoverable distortions in the ESEEM traces and the resulting spectra. The integrated RV ESEEM is free from such distortions and represents a robust practical technique for obtaining dead time- and blind spots-free spectra of nuclear transitions, without homonuclear combination lines. As an application example, the integrated RV ESEEM was used to obtain the spectrum of a strongly-coupled proton of the OH ligand of the Mo(V) active center of the low-pH form of the molybdoenzyme sulfite oxidase.     

2D-REFINE spectroscopy: separation of overlapping hyperfine spectra

We show on a mixture of three spectrally overlapping paramagnetic compounds TEMPO, BDPA and CuHis that it is possible to separate their field-swept and hyperfine spectra based on the difference in their longitudinal relaxation times T1. This was achieved in a two-dimensional experiment, where one dimension corresponds to the spectral domain and the second dimension encodes the relaxation behavior of the individual compound. Inverse Laplace Transform with respect to this domain separates the field-swept and hyperfine spectra of the individual compounds in the relaxation rate domain. This extends our formerly proposed Relaxation Filtered Hyperfine (REFINE) method to be applicable to more than two spectrally overlapping spectra by adding a further dimension to the chosen EPR experiment.     

“Unusual” lines observed in low-frequency cw ENDOR of photoexcited triplet state molecules: the primary donor triplet in photosynthetic reaction centers as an example

The origin of frequently observed “negative” (opposite phase) ENDOR lines in the low-frequency region of triplet state ENDOR spectra is explained in terms of microwave hole burning and RF modulation phenomena. From this, a new method of detecting burnt side holes in EPR spectra is derived which is based on cw ENDOR instrumentation. The method uses the modulation satellites that are induced by a longitudinal RF field component and appear around any EPR line, including burnt holes (“negative” lines). The longitudinal RF field was generated by a coil oriented parallel to the external field, but a longitudinal component of the RF field also exists in most conventional ENDOR spectrometers because of slight misalignments of the ENDOR coil generating the transversal RF field. The lines it induces in the low-frequency part of ENDOR spectra are generally considered as artifacts. It is shown, however, that RF induced modulation satellites provide valuable information concerning the lines distant from the spectral position in the EPR spectrum chosen for ENDOR observation. This allows one to record the pattern of side holes burnt by microwave saturation through forbidden transitions that carries information about ENDOR frequencies comparable to what can be extracted from ESEEM experiments. Such comparability is demonstrated for examples of nitrogen ENDOR of photoexcited triplet states of the primary donor in photosynthetic reaction centers and related compounds.     

Characteristics of ESEEM and HYSCORE spectra of S>1/2 centers in orientationally disordered systems

One- and two-dimensional electron-spin echo envelope modulation (ESEEM) spectra of Kramers’ multiplets in orientationally disordered systems are simulated using a simple mathematical model. A fairly general high-field spin Hamiltonian is considered with a general g-tensor and arbitrary relative orientations between all tensors involving the electron-spin S, the nuclear spin I, and their interaction. The zero field splitting (ZFS) and the nuclear quadrupole interactions are, however, approximated by their respective secular part in a way that retains all orientation dependencies and it is assumed that the nuclear quadrupole interaction is smaller than the hyperfine interaction. These approximations yield an effective sublevel nuclear Hamiltonian for each EPR transition and are sufficient to account for the most important characteristics of the ESEEM spectra of high electronic multiplets in orientationally disordered systems. Moreover, they allow to obtain some analytical expressions that for I=1/2 illuminate important aspects of 2D hyperfine sublevel correlation (HYSCORE) experiments in S=3/2, 5/2 systems. The pulses are considered as ideal and selective with respect to the different EPR transitions. The contributions of the latter to the echo intensity are weighed according to their different nutation angles and equilibrium Boltzmann populations. For simple axial cases with I=1/2, analytical expressions, analogous to the S=1/2 case, were derived for: (i) the modulation depth, (ii) the lineshapes of the HYSCORE cross-correlation ridges, and (iii) ENDOR powder pattern. Experimental results obtained from Mn(D₂O)₆²⁺ and VO(D₂O)₅²⁺ in frozen solutions are presented, compared, and analyzed in light of the theoretical part.     

1H one- and two-dimensional five-pulse ESEEM investigations on a radiation center in betaine phosphite single crystals

1D and 2D five-pulse ESEEM experiments on a PO ₃ ^2? center in γ-irradiated betaine phosphite are presented to study the protons bound to the radical in more detail. The ESEEM results are in accordance with previous ENDOR investigations. From the 2D fivepulse ESEEM experiments the¹H ENDOR assignment in the ferroelectric ordered state of betaine phosphite could be completed. Additionally, the occurrence of ordered proton states in the disordered paraelectric high-temperature phase and of disordered proton states in the ordered ferroelectric low-temperature phase as well could be observed due to the enhanced resolution of the 2D method.     

Nonperturbative simulation of two-pulse electron spin-echo-envelope modulation spectra. Comparison with experimental single-crystal spectra

The direct method of calculating magnetic observables provided by the Liouville formalism is utilized for a nonperturbative numerical treatment of electron spin-echo-envelope modulation (ESEEM). Calculated time-domain signals are compared with experimental ones. The particular system studied is the C0₂ anion trapped in NaHC₂O₄·H₂O single crystals. The experimental ESEEM spectra show modulations due to both proton and sodium nuclei. The hyperfine and quadrupole tensors, determined independently by ENDOR spectroscopy, are used as input for calculating the ESEEM spectra.     

Radio frequencies in EPR: Conventional and advanced use

This mini-review focuses on various aspects of the application of radio frequency (rf) irradiation in electron paramagnetic resonance (EPR). The development of the electron-nuclear double resonance (ENDOR) technique is briefly described, and we highlight the use of circularly polarized rf fields and pulse ENDOR methodology in one- and two-dimensional experiments. The capability of pulse ENDOR at Q-band is illustrated with interesting experimental examples. Electron spin echo envelope modulation effects induced by an rf field in liquid samples demonstrate another role which rf fields can play. Technical achievements in the design of ENDOR resonators are illustrated by the example of a bridged loop-gap resonator. Finally, the influence of longitudinal rf fields on the dynamics of EPR transitions is explained using a dressed spin resonance treatment.     

ESEEM of disordered systems in frequency domain: Analytical formulae in the case of nuclear spin 1/2 and weak hyperfine interaction

The analytical expressions for the spectral density of the dead time free electron spin echo envelope modulation (ESEEM) signal of disordered system are obtained for a paramagnetic center with nuclear spin 1/2 and weak axially symmetric hyperfine interaction. The spectral density is given by the Fourier transformation of the ESE signal averaged over all orientations. The order of the two linear operations may be changed. Fourier transformation of the nonaveraged ESE signal supplies us with the sum of the Dirac δ -functions. Averaging of such a spectrum is a rather trivial operation leading to the spectral densities in the final form.     

The Mo−OH proton of the low-pH form of sulfite oxidase: Comparison of the hyperfine interactions obtained from pulsed ENDOR, CW-EPR and ESEEM measurements

Pulsed electron nuclear double resonance (ENDOR) spectra have been obtained for the exchangeable Mo-OH proton of the low-pH form of native chicken liver sulfite oxidase (SO) and recombinant human SO for the first time. The spectra of the two enzymes are very similar, indicating a similar binding geometry of the hydroxyl ligand to the Mo center. The isotropic hyperfine interaction (hfi) constant for the proton of the OH ligand in both enzymes is about 26 MHz. The anisotropic components of the hfi obtained from the pulsed ENDOR spectra are about 1.6–1.8 times larger than those obtained by continuous-wave electron paramagnetic resonance and electron spin echo envelope modulation. These hfi differences are explained by a rotational disorder of the Mo-OH group. A similar rotational disorder of the coordinated exchangeable ligand has been found previously for the high-pH and phosphate-inhibited forms of SO.     

A Davies/Hahn multi-sequence for studies of spin relaxation in pulsed ENDOR

We extend earlier studies of the effects of relaxation on the intensities of pulsed ENDOR signals by introducing a Davies/Hahn (D/H) pulsed ENDOR multi-sequence that corresponds to a series of Davies sequences with the preparation pulse 'turned off'. In this pulse train, the Hahn [pi/2, pi] detection pulse pair of sequence n-1 both generates the echo detected for that sequence and acts as the preparation portion of sequence n, in effect replacing the pi preparation pulse of the Davies sequence. We show both theoretically, through a master-equation approach, and with both (1)H(I=1/2) and (14)N(I=1) ENDOR experiments on the non-heme Fe enzymes, superoxide reductase (SOR) (S=1/2) and AntDO (S=3/2), that under conditions of high electron-spin polarization (high microwave frequency/low temperature) the D/H multi-sequence allows simplification of ENDOR spectra by suppression of nuclear transitions associated with the m(S)=+1/2 (alpha) manifold. As such suppression depends on the sign of A, it allows determination of this sign. The suppression as a function of the time between individual sequences is found to exhibit behaviors that can be classified into three regimes of the ratio of cross-relaxation to spin-lattice relaxation rates: strong cross-relaxation (X-case); comparable rates (XL); negligible cross relaxation (L). Interestingly, the ENDOR behavior of the S=1/2 SOR center indicates it is an L case, while the S=3/2 AntDO is an L case. Overall, the D/H protocol appears to be a robust and general tool for using relaxation effects to manipulate ENDOR spectra.     

Electrically detected electron-spin-echo envelope modulation: a highly sensitive technique for resolving complex interface structures

We show that the electrical detection of electron-spin-echo envelope modulation (ESEEM) is a highly sensitive tool to study interfaces. Taking the Si/SiO2 interface defects in phosphorus-doped crystalline silicon as an example, we find that the main features of the observed echo modulation pattern allow us to develop a microscopic model for the dangling-bond-like P(b0) center by comparison with the results of ab initio calculations. The ESEEM spectrum is found to be far more sensitive to the defect characteristics than the spectrally resolved hyperfine splitting itself.     

A novel approach to separating EPR lines arising from species with different transition moments

In pulsed EPR, spectral contributions from several species in one sample can be separated based on different EPR transition probabilities. This is usually done by monitoring the Rabi nutations in a 2D experiment. By using long pulses, the FID and echo shapes of species with different transition probabilities differ significantly, including temporal shifts of the observed echo signals in a two-pulse ESE experiment. These shifts can be used to disentangle spectral components in a 1D field-swept ESE experiment by choosing an appropriate detection time. This approach is demonstrated by experiments on a sample containing Mn(2+) and Cr(3+) centers as well as on an exchange-coupled Mn(III)/Mn(IV) system with Mn(2+) contaminations.     

Orientation Information from the Dipolar Interaction Between a Complex in the Excited Quartet State and a Doublet Spin Label

An algorithm is proposed for deriving the position of a stable radical relative to a photoexcited quartet state from the electron spin–spin interactions measured by double resonance methods. Intersystem crossing generates multiplet polarization in the quartet state and microwave excitation of the ±3/2 ? ±1/2 transitions converts the multiplet polarization into net polarization of the ±1/2 levels. The dependence of the electron spin echo envelope modulation (ESEEM) of the +1/2 ? ?1/2 transition on the field/frequency of the stimulation pulse is demonstrated. The algorithm is tested by comparing the predicted ESEEM patterns to those from explicit numerical calculations of the spin evolution (so-called numerical experiments), which act as a model for experiment results. The comparison demonstrates that within the point-dipole approximation it is feasible to obtain not only the distance between the two paramagnetic centers but also the orientation of the distance vector relative to the principal axes of the quartet state.     

The effect of spin relaxation on ENDOR spectra recorded at high magnetic fields and low temperatures

A simple theoretical model that describes the pulsed Davies electron-nuclear double resonance (ENDOR) experiment for an electron spin S = (1/2) coupled to a nuclear spin I = (1/2) was developed to account for unusual W-band (95 GHz) ENDOR effects observed at low temperatures. This model takes into account the thermal polarization along with all internal relaxation processes in a four-level system represented by the electron- and nuclear-spin relaxation times T(1e) and T(1n), respectively, and the cross-relaxation time, T(1x). It is shown that under conditions of sufficiently high thermal spin polarization, nuclei can exhibit asymmetric ENDOR spectra in two cases: the first when t(mix) > T(1e) and T(1n), T(1x) > T(1e), where ENDOR signals from the alpha manifold are negative and those of the beta manifold positive, and the second when the cross- and/or nuclear-relaxation times are longer than the repetition time (t(mix) < T(1e) < t(R) and T(1n), T(1x) > t(R)). In that case the polarization of the ENDOR signals becomes opposite to the previous case, the lines in the alpha manifolds are positive, and those of the beta manifold are negative. This case is more likely to be encountered experimentally because it does not require a very long mixing time and is a consequence of the saturation of the nuclear transitions. Using this model the experimental t(mix) and t(R) dependencies of the W-band (1)H ENDOR amplitudes of [Cu(imidazole)(4)]Cl(2) were reproduced and the values of T(1e) and T(1x) > T(1e) were determined. The presence of asymmetry in the ENDOR spectrum is useful as it directly provides the sign of the hyperfine coupling. The presented model allows the experimentalist to adjust experimental parameters, such as t(mix) and t(R), in order to optimize the desired appearance of the spectrum.     

Self-Aggregation and Orientation of the Ion Channel-Forming Zervamicin IIA in the Membranes of ePC Vesicles Studied by cw EPR and ESEEM Spectroscopy

The ion channel-forming peptide antibiotic zervamicin A was studied in egg phosphocholin lipid membranes of large multilamellar vesicles (LMV) at 77 K. Continuous wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) methods combined with site-specific electron spin labeling were used to study the aggregation and immersion depth of two analog molecules, i.e., each monolabeled either at the N- or C-terminal end of the helical molecule. Analysis of the shape of the EPR spectra indicates that zervamicin molecules form aggregates in which the dipolar interaction between the spin labels at the N-terminus is substantially larger than that between the labels at the C-terminus. The ESEEM method was used to study the interaction between the nitroxide radical spin labels of the zervamicin molecules and deuterium nuclei in LMV, which were prepared using a D₂O buffer. It is established that the largest amplitude of deuterium modulation of the unpaired electron is observed for zervamicin molecules labeled at the N-terminus. Based on the analysis of the Fourier parameters of the deuterium modulated spectrum, a model of the immersion depth of the terminal ends of the zervamicin molecule in a lipid bilayer is formulated. All of the spin labels at the N-terminus are grouped at the lipid–water interface, whereas 60% of labels at the C-terminus are located at the lipid–water interface and 40% are more deeply inserted into the lipid bilayer.