ENDOR double resonance

B1.15.5.1 ELECTRON-NUCLEAR DOUBLE RESONANCE SPECTROSCOPY (ENDOR)... 

This chapter concludes with a brief description of one advanced technique, Electron Nuclear Double Resonance (ENDOR), the capabilities for which, unlike pulsed methods, may be added as a relatively minor modification to commercial CW ESR spectrometers. 

Double resonance techniques, on the other hand, of which the earliest was ENDOR (described in Chapter 1), have greatly benefited from advances in signal processing technology, of the sort now employed, for example, in wireless communication systems. 

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... 

As discussed in Chapter 6, in systems with more than one unpaired electron the ESR spectrum contains features that involve electron-electron coupling parameters analogous to the nuclear hyperfine parameters. In those types of samples the advantages of double resonance are carried out by employing the use of two different microwave frequencies, one fixed and saturating, and one variable frequency that searches for transitions. This technique is known as ELDOR (electron-electron double resonance).38,40,41,44 It has been used much less than ENDOR and usually requires custom-built equipment. 

A major limitation of CW double resonance methods is the sensitivity of the intensities of the transitions to the relative rates of spin relaxation processes. For that reason the peak intensities often convey little quantitative information about the numbers of spins involved and, in extreme cases, may be undetectable. This limitation can be especially severe for liquid samples where several relaxation pathways may have about the same rates. The situation is somewhat better in solids, especially at low temperatures, where some pathways are effectively frozen out. Fortunately, fewer limitations occur when pulsed radio and microwave fields are employed. In that case one can better adapt the excitation and detection timing to the rates of relaxation that are intrinsic to the sample.50 There are now several versions of pulsed ENDOR and other double resonance methods. Some of these methods also make it possible to separate in the time domain overlapping transitions that have different relaxation behavior, thereby improving the resolution of the spectrum. 

M. Bennati, C.T. Farrar, J.A. Bryant, S.J. Inati, V. Weis, G.J. Gerfen, P. Riggs-Gelasco, J. Stubbe and R.G. Griffin, Pulsed electron-nuclear double resonance (ENDOR) at 140 GHz, J. Magn. Reson., 1999, 138, 232. 

The development of a wide range of special forms of EPR was initiated when the idea of double resonance (using simultaneous irradiation by two different sources) was cast in 1956 by G. Feher at Bell Telephone Labs in his seminal paper on ENDOR, electron nuclear double resonance (Feher 1956). BioEPR applications of ENDOR were later developed on flavoprotein radicals in a collaboration of A. Ehrenberg and L. E. G. Eriksson in Stockholm, Sweden, and J. S. Hyde at Varian in Palo Alto, California (Ehrenberg et al. 1968), and on metalloproteins in a joint effort of the groups of R. H. Sands in Ann Arbor, I. C. Gunsalus in Urbana, Illinois, and H. Beinert in Madison (Fritz et al. 1971). 

Double-resonance spectroscopy involves the use of two different sources of radiation. In the context of EPR, these usually are a microwave and a radiowave or (less common) a microwave and another microwave. The two combinations were originally called ENDOR (electron nuclear double resonance) and ELDOR (electron electron double resonance), but the development of many variations on this theme has led to a wide spectrum of derived techniques and associated acronyms, such as ESEEM (electron spin echo envelope modulation), which is a pulsed variant of ENDOR, or DEER (double electron electron spin resonance), which is a pulsed variant of ELDOR. The basic principle involves the saturation (partially or wholly) of an EPR absorption and the subsequent transfer of spin energy to a different absorption by means of the second radiation, leading to the detection of the difference signal. The requirement of saturability implies operation at close to liquid helium, or even lower, temperatures, which, combined with long experimentation times, produces a... 

Fritz, J., Anderson, R., Fee, J., Palmer, G, Sands, R.H., Tsibris, J.C.M., Gunsalus, I.C., Orme-Johnson, W.H., and Beinert, H. 1971. The iron electron-nuclear double resonance (ENDOR) of two-iron ferredoxins from spinach, parsley, pig adrenal cortex and Pseudomonas putida. Biochimica et Biophysica Acta 253 110-133. 

As most of the nitroxyl spin-labelled synthetic derivatives of conjugated polyenes are light yellow crystals, the bond lengths were determined by X-ray crystallography38. The spectroscopic method used to measure the conformation is electron nuclear double resonance (ENDOR). It is beyond the scope of the present review to explain the method38 but the authors of the pertinent paper conclude that ENDOR is an accurate non-crystallographic method to determine polyene structures in solution. 

ESR and ENDOR (electron-nucleus double resonance) spectroscopies110. 

Since the phenoxyls possess an S = ground state, they have been carefully studied by electron paramagnetic spectroscopy (EPR) and related techniques such as electron nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM). These powerful and very sensitive techniques are ideally suited to study the occurrence of tyrosyl radicals in a protein matrix (1, 27-30). Careful analysis of the experimental data (hyperfine coupling constants) provides experimental spin densities at a high level of precision and, in addition, the positions of these tyrosyls relative to other neighboring groups in the protein matrix. 

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