Continuous wave ENDOR

Electron-nuclear double resonance (ENDOR) spectroscopy A magnetic resonance spectroscopic technique for the determination of hyperfine interactions between electrons and nuclear spins. There are two principal techniques. In continuous-wave ENDOR the intensity of an electron paramagnetic resonance signal, partially saturated with microwave power, is measured as radio frequency is applied. In pulsed ENDOR the radio frequency is applied as pulses and the EPR signal is detected as a spin-echo. In each case an enhancement of the EPR signal is observed when the radiofrequency is in resonance with the coupled nuclei. 

ENDOR spectra for positive ion radicals obtained by UV irradiation of [3-carotene (1) and canthaxanthin (16) were discussed [125]. Improved techniques using continuous wave ENDOR (CW ENDOR) including ID and 2D ESEEM techniques for cation radicals of canthaxanthin (16) and of an apocarotenal were recently reported [133],... 

This limitation is not there for pulsed ENDOR methods, which can be used at all microwave frequencies. In ENDOR, the sample is irradiated with a combination of microwaves and radio waves. Continuous-wave ENDOR was already introduced in 1956 by Feher [1] and for a long time remained an important tool to determine the hyperfine and nuclear quadrupole interactions. However, nowadays this technique is largely replaced by the pulsed counterparts, which are more versatile. Two of the most commonly used ENDOR pulse sequences are Davies ENDOR [18] and Mims ENDOR [ 19]. In these techniques a combination of microwave pulses and a n radio frequency (RF) pulse with variable RF is used. A first set of microwave pulses creates electron polarization. When the RF matches one of the nuclear transitions, the populations of the different energy levels will be affected. This will change the electron polarization that is read out by a last sequence of microwave pulses, usually via electron spin echo detection as a function of the radio frequency. In this way, the nuclear frequencies can be directly detected. 

Grupp A and Mehring M 1990 Pulsed ENDOR spectroscopy in solids Modern Pulsed and Continuous-Wave Electron Spin Resonance ed L Kevan and M K Bowman (New York Wiley) ch 4, pp 195-229... 

CW, continuous wave Cys, cysteine DFT, density functional theory ENDOR, electron nuclear double resonance ehba, 2-ethyl-2-hydroxybutanoate2 EPR, electron paramagnetic resonance Glc6P, D-glucose 6-phosphate GSH, reduced glutathione HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid Hex, aldohexose ... 

As mentioned above, ENDOR is the detection of NMR resonances by observing their effect on an EPR signal, whether in continuous wave (CW) or pulsed mode. The NMR... 

The following sections provide a more detailed description of the hyperfine interaction as measured by ENDOR spectroscopy, a description of ENDOR instrumentation, and the types of ENDOR experiments that can be performed. Finally, examples of the application of ENDOR spectroscopy to a variety of biomolecules are described. In this brief review many statements are made without reference for details the reader is referred to the variety of more extensive works for the theory of EPR and hyperfine interactions and reviews of applications of continuous wave (cw) and pulsed ENDOR and ESEEM (electron spin echo envelope modulation) techniques. ... 

Morrissey SR, Horton TE, DeRose VJ (2000) Mn sites in the hammerhead ribozyme investigated by EPR and continuous-wave Q-band ENDOR spectroscopies. J Am Chem Soc 122(14) 3473-3481... 

Electron paramagnetic resonance (EPR) spectroscopy [1-3] is the most selective, best resolved, and a highly sensitive spectroscopy for the characterization of species that contain unpaired electrons. After the first experiments by Zavoisky in 1944 [4] mainly continuous-wave (CW) techniques in the X-band frequency range (9-10 GHz) were developed and applied to organic free radicals, transition metal complexes, and rare earth ions. Many of these applications were related to reaction mechanisms and catalysis, as species with unpaired electrons are inherently unstable and thus reactive. This period culminated in the 1970s, when CW EPR had become a routine technique in these fields. The best resolution for the hyperfine couplings between the unaired electron and nuclei in the vicinity was obtained with CW electron nuclear double resonance (ENDOR) techniques [5]. 

Electron spin resonance (ESR) spectroscopy is a very powerful and sensitive method for the characterization of the electronic structures of materials with unpaired electrons. There is a variety of ESR techniques, each with its own advantages. In continuous wave ESR (CW-ESR), the sample is subjected to a continuous beam of microwave irradiation of fixed frequency and the magnetic field is swept. Different microwave frequencies may be used and they are denoted as S-band (3.5 GHz),X-band (9.25 GHz), K-band (20 GHz), Q-band (35 GHz) and W-band (95 GHz). Other techniques, such as electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, record in essence the NMR spectra of paramagnetic species. 

H. Kurreck, B. Kirste, W. Lubitz, ENDOR Spectroscopy of Radicals in Solution, VCH, 1988 E. R. Davies, Phys. Lett. A 1974, 47, 1-2 Arthur Grupp and Michael Mehring, in Modem Pulsed and Continuous Wave ESR , 1979, 195-229. 

Abstract Multi-resonance involves ENDOR, TRIPLE and ELDOR in continuous-wave (CW) and pulsed modes. ENDOR is mainly used to increase the spectral resolution of weak hyperfine couplings (hfc). TRIPLE provides a method to determine the signs of the hfc. The ELDOR method uses two microwave (MW) frequencies to obtain distances between specific spin-labeled sites in pulsed experiments, PELDOR or DEER. The electron-spin-echo (ESE) technique involves radiation with two or more MW pulses. The electron-spin-echo-envelope-modulation (ESEEM) method is particularly used to resolve weak anisotropic hfc in disordered solids. HYSCORE (Hyperfine Sublevel Correlation Spectroscopy) is the most common two-dimensional ESEEM method to measure weak hfc after Fourier transformation of the echo decay signal. The ESEEM and HYSCORE methods are not applicable to liquid samples, in which case the FID (free induction decay) method finds some use. Pulsed ESR is also used to measure magnetic relaxation in a more direct way than with CW ESR. 

Multi-resonance and pulsed ESR techniques can provide better spectral resolution than conventional ESR. Multi-resonance involves ENDOR, TRIPLE and ELDOR. In an ENDOR experiment a radiofrequency (RF) field is applied in addition to the microwave (MW) employed in standard continuous wave (CW) ESR. ENDOR is mainly used to increase the spectral resolution, so that overlapping or unresolved hyperfine structure in the ESR spectra can be detected. In the classical work by Feher [1] the radiofrequency was continuously swept. CW X-band spectrometers with an ENDOR attachment have been commercially available for a long time. Accessories for other frequency bands and for pulsed ESR have been developed more recently. In a TRIPLE experiment two RE fields are applied [2, 3]. A theoretical application has been to determine the relative signs of two hyperfine couplings. In an ELDOR experiment two MW frequencies are applied. Early applications using... 

Most of the specialized techniques involve the use of fairly sophisticated instrumentation these can be divided roughly into two categories continuous wave (CW) and pulse methods. Electron-nuclear double resonance (ENDOR) and ESR imaging will be discussed in the CW category, while a variety of... 

Figure 1 First-derivative spectra obtained by continuous-wave techniques for the phenalenyl radical (A) ESR, (B) ENDOR, (C) special TRIPLE resonance, (D) general TRIPLE resonance, and (E) ENDOR-induced ESR. The arrows indicate the positions of the RF pumping frequencies there are several possibilities in (D). The RF pumping frequency in (E) is set in the same position as shown in (B). (Reprinted from Kurreck H, Kirste B, and Lubitz W (1988) Electron Nuclear Double Resonance Spectroscopy of Radicals In Solution. Weinheim VCH Publishers.)...

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