Single-crystal (57)Fe Q-band ENDOR study of the 4 iron-4 sulfur cluster in its reduced [4Fe-4S](1+) state.

(57)Fe Q-band ENDOR has been used to study the [4Fe-4S](1+) state created by gamma irradiation of single crystals of the synthetic model compound [N(C(2)H(5))(4)](2)[Fe(4)S(4)(SCH(2)C(6)H(5))(4)] enriched in (57)Fe. This compound is an excellent biomimetic model of the active sites of many 4 iron-4 sulfur proteins, enabling detailed and systematic studies of its oxidized [4Fe-4S](3+) and reduced [4Fe-4S](1+) paramagnetic states. Taking advantage of the fact that Q-band ENDOR, in contrast with X-Band ENDOR, allows for a very good separation of the (57)Fe transitions from those of the protons, the complete hyperfine tensors of the four iron atoms for the [4Fe-4S](1+) species has been measured with precision. For each iron atom, the electron orbital and electron spin isotropic contributions have been determined separately. Moreover, it is remarkable that two (57)Fe hyperfine tensors attributed to the ferrous pair of iron atoms are very different. In effect, one tensor presents a much larger anisotropic part and a much smaller isotropic part than those of the other. This difference has been interpreted in terms of a differential electron orbital hyperfine interaction among the two ferrous ions.     

Determination of signs of hyperfine coupling constants for an S = ½ system through E.P.R., ENDOR and double ENDOR

A systematic method of obtaining relative signs of hyperfine coupling constants is described. It applies to systems consisting of (a) a set of one or more nuclei coupled fairly strongly to the electron spin, and possessing a two-fold (or higher) axis of symmetry, together with (b) a set of weakly coupled nuclei defining superhyperfine transitions. ENDOR measurements for several E.P.R. hyperfine transitions, with the field oriented along the symmetry axis, give relative signs of hyperfine components for this direction. Signs for the other directions can then be obtained through ENDOR measurements on a single hyperfine transition at various field orientations. Additional double ENDOR measurements may be necessary for very weakly coupled nuclei. This method can complement double ENDOR studies in favourable cases. It is illustrated by the determination of signs of coupling constants of protons and of ⁷⁵As in the AsO₄ ^4- radical in KH₂AsO₄.     

Single-Crystal Fe Q-Band ENDOR Study of the 4 Iron–4 Sulfur Cluster in its Reduced [4Fe–4S] State

⁵⁷Fe Q-band ENDOR has been used to study the [4Fe–4S]¹⁺ state created by γ irradiation of single crystals of the synthetic model compound [N(C₂H₅)₄]₂[Fe₄S₄(SCH₂C₆H₅)₄] enriched in ⁵⁷Fe. This compound is an excellent biomimetic model of the active sites of many 4 iron–4 sulfur proteins, enabling detailed and systematic studies of its oxidized [4Fe–4S]³⁺ and reduced [4Fe–4S]¹⁺ paramagnetic states. Taking advantage of the fact that Q-band ENDOR, in contrast with X-Band ENDOR, allows for a very good separation of the ⁵⁷Fe transitions from those of the protons, the complete hyperfine tensors of the four iron atoms for the [4Fe–4S]¹⁺ species has been measured with precision. For each iron atom, the electron orbital and electron spin isotropic contributions have been determined separately. Moreover, it is remarkable that two ⁵⁷Fe hyperfine tensors attributed to the ferrous pair of iron atoms are very different. In effect, one tensor presents a much larger anisotropic part and a much smaller isotropic part than those of the other. This difference has been interpreted in terms of a differential electron orbital hyperfine interaction among the two ferrous ions.     

General formulae for hyperfine structure and endor frequencies

General expressions for hyperfine (superhyperfine) structure and ENDOR frequencies are derived. It is only assumed that the hyperfine (superhyperfine) interaction is much smaller than the electronic spin transition energy but the electronic spin-Hamiltonian may be of arbitrary form. To obtain the ENDOR frequencies one need only know the electron spin matrix elements. These influence the direction and magnitude of the effective magnetic field which act on the nucleus.     

Nuclear relaxation effects in Davies ENDOR variants

A recent article by Yang and Hoffman [T.-C. Yang, B.M. Hoffman, J. Magn. Reson., 181 (2006) 280] presents a ‘Davies/Hahn ENDOR multi-sequence’ in which the α and β peaks of an electron-nuclear double resonance (ENDOR) spectrum can be distinguished. This represents one instance of a family of ENDOR sequences which have no initial microwave inversion pulse, and which can reveal information about nuclear relaxation rates and the signs of hyperfine coupling constants. Here we discuss the more general set of such sequences, which we refer to as Saturated Pulsed ENDOR, and show how signal sensitivity can be optimised within the context of this new technique. Through simulations, we compare its performance to other techniques based on Davies ENDOR, and experimentally illustrate its properties using the non-heme Fe enzyme anthranilate dioxygenase AntDO. Finally, we suggest a protocol for extracting both the magnitude and sign of the hyperfine tensor using a combination of ENDOR techniques.     

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.     

Schonland ambiguity in the electron nuclear double resonance analysis of hyperfine interactions: principles and practice

For the analysis of the angular dependence of electron paramagnetic resonance (EPR) spectra of low-symmetry centres with S=1/2 in three independent planes, it is well-established-but often overlooked-that an ambiguity may arise in the best-fit g<--> tensor result. We investigate here whether a corresponding ambiguity also arises when determining the hyperfine coupling (HFC) A<--> tensor for nuclei with I=1/2 from angular dependent electron nuclear double resonance (ENDOR) measurements. It is shown via a perturbation treatment that for each set of M(S) ENDOR branches two best-fit A<--> tensors can be derived, but in general only one unique solution simultaneously fits both. The ambiguity thus only arises when experimental data of only one M(S) multiplet are used in analysis or in certain limiting cases. It is important to realise that the ambiguity occurs in the ENDOR frequencies and therefore the other best-fit result for an ENDOR determined A<--> tensor depends on various details of the ENDOR experiment: the M(S) state of the fitted transitions, the microwave frequency (or static magnetic field) in the ENDOR measurements and the rotation planes in which data have been collected. The results are of particular importance in the identification of radicals based on comparison of theoretical predictions of HFCs with published literature data. A procedure for obtaining the other best-fit result for an ENDOR determined A<--> tensor is outlined.     

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.     

Non-kramers ENDOR and ESEEM of the S = 2 ferrous ion of [Fe(II)EDTA](2-)

We report here the first non-Kramers (NK) ESEEM and ENDOR study of a mononuclear NK center, presenting extensive parallel-mode ESEEM and ENDOR measurements on the S(t) = 2 ferrous center of [Fe(II)ethylenediamine-N,N,N',N'-tetraacetato](2-); [Fe(II)EDTA)](2-). The results disclose an anomalous equivalence of the experimental patterns produced by the two techniques. A simple theoretical treatment of the frequency-domain patterns expected for NK-ESEEM and NK-ENDOR rationalizes this correspondence and further suggests that the very observation of NK-ENDOR is the result of an unprecedentedly large hyperfine enhancement effect. The mixed nitrogen-carboxylato oxygen coordination of [Fe(II)EDTA](2-) models that of the protein-bound diiron centers, although with a higher coordination number. Analysis of the NK-ESEEM measurements yields the quadrupole parameters for the (14)N ligands of [Fe(II)EDTA](2-), K = 1.16(1) MHz, 0 相似文献   

Mössbauer study of 57Fe in GaAs and GaP following 57Mn+ implantation

Ion implantation provides a precise method of incorporating dopant atoms in semiconductors, provided lattice damage due to the implantation process can be annealed and the dopant atoms located on regular lattice sites. We have undertaken ⁵⁷Fe emission Mössbauer spectroscopy measurements on GaAs and GaP single crystals following implantation of radioactive ⁵⁷Mn^?+? ions, to study the lattice sites of the implanted ions, the annealing of implantation induced damage and impurity–vacancy complexes formed. The Mössbauer spectra were analyzed with four spectral components: an asymmetric doublet (D1) attributed to Fe atoms in distorted environments due to implantation damage, two single lines, S1 assigned to Fe on substitutional Ga sites, and S2 to Fe on interstitial sites, and a low intensity symmetric doublet (D2) assigned to impurity–vacancy complexes. The variations in the extracted hyperfine parameters of D1 for both materials at high temperatures (T?> 400 K) suggests changes in the immediate environment of the Fe impurity atoms and different bonding mechanism to the Mössbauer probe atom. The results show that the annealing of the radiation induced damage is more prominent in GaAs compared to GaP.     

ENDOR spectroscopic and molecular orbital study of the dynamical properties of the side chain in radical anions of ubiquinones Q-1, Q-2, Q-6, and Q-10

The dynamics of the side chain of the radical anions of ubiquinones Q-1 (2,3-dimethoxy-5-methyl-6-[3-methyl-2-butenyl]-1,4-benzoquinone), Q-2, Q-6, and Q-10 have been investigated using electron nuclear double-resonance (ENDOR) spectroscopy. When radicals are produced in the liquid phase, secondary radicals are also formed. The EPR spectra of these additional radicals overlap with the radical of interest. ENDOR spectroscopy was found to be capable for studying the dynamical properties of such conditions. The temperature dependence of the isotropic hyperfine coupling constants of the beta- and gamma-protons of the side chain was measured. The activation energy of the rotation and other dynamical properties of the side chain were calculated assuming that rotation can be modeled by the classical two-jump model. The rotation energy barrier for Q-1 was also determined by the hybrid Hartree-Fock/density functional method UB3LYP with the 6-31G(d) basis set. Calculated results were in good agreement with the experimental results. Despite the numerous parameters affecting the ENDOR linewidth ENDOR spectroscopy was shown to be a potential method for studying the dynamical properties of the mixtures of the radicals. Prominent forbidden transitions appear in the ENDOR spectra when alkali ions are present in the sample. From these transitions measured ENDOR-induced EPR spectra showed an additional doublet and phase transition in electron Zeeman frequency.     

Quantitative characterization of the Mn2+ complexes of ADP and ATPγS by W-band ENDOR

W-band (95 GHz) pulsed electron nuclear double resonance (ENDOR) measurements were carried out to determine quantitatively the first coordination shell of Mn²⁺ with ADP and ATPγS. The intensity of the ENDOR effect was used for counting the number of equivalent phosphate oxygens and water ligands. Titration curves for determining the binding constant of Mn²⁺. ADP were obtained using the intensity of the X-band EPR spectrum and the³¹P ENDOR effect. Both curves gave the same binding constant showing that phosphate ligand counting is plausible, provided that an appropriate reference is available. The comparison of the³¹P ENDOR effect of the 1:1 ADP and ATPγS complexes shows that two phosphates are coordinated in both; while in ADP they are equivalent, in ATPγS they are slightly different. The reference system for water ligand counting was Mn(H₂O) ₆ ²⁺ in a H₂O-D₂O mixture. The results show a smaller error for the²H ENDOR effect, compared to the¹H ENDOR effect. Unlike the³¹P ENDOR effect, the¹H ENDOR effect dependence on [ADP] in the titration experiments showed that it is sensitive to variations in the zero-field splitting, which in turn alters the contributions of transitions other than the ‖?1/2>?‖1/2>. This results in a larger error in the determination of the number of water ligands.     

High-field ENDOR and the sign of the hyperfine coupling

Two different approaches for assigning electron nuclear double resonance (ENDOR) signals to their respectiveM _s manifolds by a controlled generation of asymmetric ENDOR spectra, are described and applied to a number of systems. This assignment then allows a straightforward determination of the sign of the hyperfine coupling. Both approaches rely on a high thermal polarization that is easily achieved at high fields and low temperatures. For high-spin systems, such asS = 5/2 the assignment is afforded by the selective inversion of the | ?3/2〉 → | ?1/2〉 electron paramagnetic resonance (EPR) transition which is highly populated as compared to its symmetric counterpart, the |1/2〉 → |3/2〉 EPR transition, and therefore is easily identified. ForS = 1/2 the determination of the sign of the hyperfine coupling becomes possible when the cross- and nuclear-spin relaxation rates are much slower than the electron-spin relaxation rate and variable mixing time pulse ENDOR is used to measure the spectrum. Under these conditions the signals of theM _s = 1/2 (α) manifold become negative when the mixing time is on order of the electron-spin relaxation time, whereas those of theM _s =?1/2 (β) manifold remain positive. Under partial saturation of the nuclear transitions and short mixing time the opposite behavior is observed. Pulse W-band¹H ENDOR experiments demonstrating these approaches were applied and the signs of the hyperfine couplings of the water ligands in Mn(H₂O) ₆ ²⁺ , the H_α and H_β histidine protons in the Cu(histidine)₂ complex, the imidazole protons in Cu(imidazole) ₄ ²⁺ and the cysteine β-protons in nitrous oxide reductase were determined.     

Doppler-free two-photon laser spectroscopy of barium I: Hyperfine splitting and isotope shift of high lying levels

The Doppler-free two-photon absorption method performed with a narrowbandc w dye laser permitted high resolution measurements of transitions from the Ba I ground state 6s ^{2 1} S ₀ to several highly excited states. The lifetimes and hyperfine splittings of these states as well as the isotope shifts of the transitions have been determined accurately. The lifetime values are in agreement with transition probability data; the hyperfine splitting results show considerable configuration interaction effects. A detailed discussion of the isotope shifts is given.     

Rubidium and proton ENDOR on ion pairs in solution

The E.S.R. spectrum of the o-dimesitoylbenzene anion-alkali cation radical shows unusually large isotropic alkali hyperfine splitting constants. We report a solution ENDOR study of this radical in which both alkali (^85,87Rb) and proton ENDOR spectra were recorded. Both the alkali and proton intensities showed a strong dependence on the metal ion nuclear spin quantum number of the E.S.R. line being saturated. This dependence is attributed to strong flip-flop cross-relaxation induced by modulation of the isotropic alkali hyperfine splitting. The powder E.S.R. spectrum of the complex reveals a small anisotropy of the Rb hyperfine splitting tensor. This indicates a small metal non-s-contribution to the half-filled molecular orbital, which is consistent with the observed relaxation behaviour and the small g shift. The intensity variations in the alkali and proton ENDOR spectra were used to determine the relative signs of all hyperfine splitting constants, and the absolute signs of the hyperfine splitting constants are deduced from a model of the structure of the complex.     

ESR study of exchange coupled pairs in copper diethyldithiocarbamate—explanation of the low temperature hyperfine anomaly

A study of the hyperfine interaction in the ESR of Cu-Cu pairs in single crystals of copper diethyldithiocarbamate as a function of temperature has shown distinct differences in the hyperfine structure in the two fine structure transitions at 20 K, the spectrum not having the same hyperfine intensity pattern in the low field fine structure transition in contrast to that of the high field transition. The details of the structure of both the fine structure transitions in the 20 K spectrum have now been explained by recognizing the fact that the mixing of the nuclear spin states caused by the anisotropic hyperfine interaction affects the electron spin states | + 1 > and | −> differently. This has incidentally led to a determination of the sign ofD confirming the earlier model. The anomalous hyperfine structure is found to become symmetric at 77 K and 300 K. It is proposed that the reason for this lies in the dynamics of spin-lattice interaction which limits the lifetime of the spin states in each of the electronic levels | − 1 >, | 0 > and | + 1 > The estimate of spin-lattice relaxation time agrees with those indicated from other studies. The model proposed here for the hyperfine interaction of pairs in the electronic triplet state is of general validity.     

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.     

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.     

Effet de second ordre pour des protons de méthylène en ENDOR

An indirect second-order effect occurs between the methylene-group protons of a radical created by gamma irradiation in a 1, 2, 4-triazol single crystal; this gives a non-crossing phenomenon which is observed on the ENDOR transitions. It is necessary to take this into account in the expression for the transitions, which then allows us to determine the hyperfine tensors of the two protons. Moreover, the presence of this effect helps in the identification of the transitions and the signs of the couplings.     

Pulsed ENDOR Spectroscopy at Large Thermal Spin Polarizations and the Absolute Sign of the Hyperfine Interaction

It is shown that in pulsed Mims-type ENDOR experiments performed at 95 GHz and 1.2 K the sign of the ENDOR signal can be positive, corresponding to an increase of the stimulated echo intensity, as well as negative, corresponding to a decrease of the stimulated echo. The positive “anomalous” sign is not observed at conventional EPR frequencies. It is explained that the effect arises through spin–lattice relaxation in the situation of large thermal spin polarizations and that it allows the determination of the absolute sign of the hyperfine interaction.