ENDOR experiments

The complete spin Hamiltonian for a description of EPR and ENDOR experiments is given by... 

A refinement of the ENDOR experiment is electron-nnclear-nnclear triple resonance, now commonly denoted TRIPLE. In TRIPLE experiments one monitors the effect of a simnltaneons excitation of two nnclear spm transitions on the level of the EPR absorption. Two versions, known as special TRIPLE (ST) and general TRIPLE (GT), are rontinely perfonned on connnercially available spectrometers. 

Muns ENDOR mvolves observation of the stimulated echo intensity as a fimction of the frequency of an RE Ti-pulse applied between tlie second and third MW pulse. In contrast to the Davies ENDOR experiment, the Mims-ENDOR sequence does not require selective MW pulses. For a detailed description of the polarization transfer in a Mims-type experiment the reader is referred to the literature [43]. Just as with three-pulse ESEEM, blind spots can occur in ENDOR spectra measured using Muns method. To avoid the possibility of missing lines it is therefore essential to repeat the experiment with different values of the pulse spacing Detection of the echo intensity as a fimction of the RE frequency and x yields a real two-dimensional experiment. An FT of the x-domain will yield cross-peaks in the 2D-FT-ENDOR spectrum which correlate different ENDOR transitions belonging to the same nucleus. One advantage of Mims ENDOR over Davies ENDOR is its larger echo intensity because more spins due to the nonselective excitation are involved in the fomiation of the echo. 

Figure 2.13 Example of an ENDOR experiment for a single spin-1/2 nucleus. See text for explanation.

In Chapter 2, ENDOR (electron-nuclear double resonance) was briefly described. To perform an ENDOR experiment it is necessary to apply both a radiofrequency and a microwave frequency, effectively performing simultaneous NMR and ESR, respectively, on the sample. The experiment is performed at a fixed magnetic field, with the ESR saturating frequency centered on a... 

Finally, the use of DNP of shallow donors to enhance both 67Zn and surface ft nuclear polarizations has been demonstrated in ZnO nanoparticles by observation of EPR features rather than direct NMR observation [85, 87]. The electronic wavefunctions of these donors in ZnO have been probed by ENDOR experiments [36, 97], There is much potential for directly observing NMR with the sensitivity greatly enhanced by DNP not only in ZnO but in other nanoparticles as well. 

As mentioned above, in an ENDOR experiment the rf field is swept while the static magnetic field is held at a constant position in the EPR spectrum. For slow sweep rates and narrow EPR lines a device would be desirable which is able to stabilize the ratio of the microwave frequency to the static magnetic field. The applicaiton of a commercially available field/frequency lock system is restricted to a region of 6 mT about the DPPH resonance field33). In metal complexes with strongly anisotropic EPR spectra, however,... 

In polarization modulated ENDOR spectroscopy (PM-ENDOR)45, discussed in Sect. 4.7, the linearly polarized rf field B2 rotates in the laboratory xy-plane at a frequency fr fm, where fm denotes the modulation frequency of the rf carrier. In a PM-ENDOR experiment the same type of cavity, with two rf fields perpendicular to each other, and the same rf level and phase control units used in CP-ENDOR can be utilized. To obtain a rotating, linearly polarized rf field with a constant magnitude B2 and a constant angular velocity Q = 2 fr (fr typically 30-100 Hz), double sideband modulation with a suppressed carrier is applied to both rf signals. With this kind of modulation the phase of the carrier in each channel is switched by 180° for sinQt = 0. In addition, the phases of the two low-frequency envelopes have to be shifted by 90° with respect to each other. The coding of the two rf signals is shown in Fig. 8. 

In this section we will be concerned with various techniques which supplement the conventional ENDOR experiment. The principles of the different methods as well as their advantages, limitations and applications will be discussed. 

During the last few years the versatility of ENDOR spectroscopy has been improved by a number of new techniques which make use either of special types of pumping fields (CP-ENDOR, PM-ENDOR), of more than one rf field (DOUBLE ENDOR, multiple quantum transitions, nuclear spin decoupling) or a different display of the spectrum (EI-EPR). In addition to these techniques, alternative methods have been developed (electron spin echo and electron spin echo ENDOR) which are able to supplement or to replace the ENDOR experiment under certain conditions. The utility of all these various advanced techniques, particularly in studies of transition metal compounds, has recently been demonstrated. 

The spin distribution in the lower homologue [15 w = 2] was most elegantly studied by Takui et al. (1989) by means of a combination of ENDOR experiments and theoretical calculations within the framework of a generalized UHF Hubbard model (Teki et al., 1987a) and a Heisenberg model (Teki et al., 1987b). The spin distribution obtained is just as expected qualitatively in Fig. 7. 

Fig. 19 n-Spin-density distribution in diphenylcarbene [15 m= ] determined by ENDOR experiments and MO calculations. The o-spin density at the divalent carbon is in parentheses. 

V7 EPR/ENDOR experiments on [NiFe] hydrogenase single crystals from D. vulgaris Miyazaki F. 

ENDOR experiments can be performed in liquid solution, in which only the isotropic hfc s (Ajso) are detected. They are proportional to the spin density at the respective nucleus. Erom the assigned isotropic hfc s a map of the spin density distribution over the molecule can be obtained. In frozen solutions and powders the anisotropic hf interactions can also be determined. Eurthermore, the method allows the detection of nuclear quadrupole couplings for nuclei with 1 1. For dominant g anisotropy as found in many metal complexes the external magnetic field can be set to several specific g values in the EPR, thereby selecting only those molecules that have their g tensor axis along the chosen field direction. In such orientation-selected spectra only those hf components are selected that correspond to this molecular orientation ( single crystal-like ENDOR ). 

Hoffman and collaborators (Doan et al. 1994) also performed ENDOR experiments on the oxidized [3Fe-4S] cluster in D. gigas hydrogenase. The authors detected resonances from strongly coupled protons which were assigned to the [3-CH2 of the cysteines and exchangeable protons that are probably involved in three different hydrogen bonds to the sulfurs of the cluster. Based on these data a model for the binding of the cluster to the protein was developed. 

In this respect the possible presence of electron spin density at the iron in the Ni-Fe cluster is of importance. This question has been answered by ENDOR experiments performed on Fe enriched [NiFe] hydrogenase (Huyett et al. 1997). In this study no significant Fe hfc was detected in the NiA, NiB and NiC states (A57p s 1 MHz). This shows that in all relevant states of the enzyme the iron is in the low-spin Fe state. 

Amazingly, low-temperature CW EPR has only been sparsely applied for the study of oxo-Cr(V) complexes,29,52-55 and even fewer studies involve CW ENDOR55,56 or pulsed EPR/ENDOR experiments,52 or attempt a combination of EPR and quantum-chemical computations.29,52,54 A great potential evidently still exists for further research along this line. 

Several recent papers have reported Density Functional Theory (DFT) calculations on the primary oxidation and reduction products observed in irradiated single crystals of the common nucleobases thymine [53], cytosine [54], guanine [55], and adenine [56]. The theoretical calculations include estimates of spin densities and isotropic and anisotropic hyperfine couplings which can be compared with experimental results (obtained from detailed EPR/ENDOR experiments). 

This review has looked at the direct effects of ionizing radiation on nucleic acids. The first step was to review detailed EPR/ENDOR experiments on irradiated model compounds at low temperatures in order to study the primary radiation-induced defects. Next, it was shown how these EPR spectra are used to simulate the EPR spectra of the DNA polymer. 

The calculation of magnetic parameters such as the hyperfine coupling constants and g-factors for oligonuclear clusters is of fundamental importance as a tool for the evaluation of spectroscopic data from EPR and ENDOR experiments. The hyperfine interaction is experimentally interpreted with the spin Hamiltonian (SH) H = S - A-1, where S is the fictitious, electron spin operator related to the ground state of the cluster, A is the hyperfine tensor, and I is the nuclear spin operator. Consequently, it is... 

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