ESEEM and ENDOR Spectroscopies

Left two-pulse [(a) primary ESEEM] and three-pulse [(b) stimulated echo ESEEM] sequences t is the (fixed) delay time between pulses one and two and T is a variable delay time. Right frequency domain and time domain (inset) of the two-pulse EESEM spectrum of VO - vanabin, recorded at the m = — 1 /2 line, at 77 K and a pulse width of 20 ns.P l The superhyperfine coupling constant = 4.5 MHz (obtained from the N double-quantum lines at 3.9 and 7.1 MHz) is in accord with amine nitrogen provided by lysines of the vanadium-binding protein. The spin echo due to proton coupling, at 13.7 MHz, was also observed. Reproduced from K. Eukui et al., J. Am. Chem. Soc. 125, 6352-6353. Copyright (2003), with permission from the American Chemical Society. 

A comparatively recent new development in ENDOR spectroscopy is electron spin echo ENDOR (ESE-ENDOR), where nuclear spin transitions are detected by their effect on a transient EPR signal (the spin echo) generated by a two- or three-pulse excitation. 

---

N.D. Chasteen and P.A. Snetsinger, ESEEM and ENDOR spectroscopy, in Physical Methods in Bioinorganic Chemistry, Spectroscopy and Magnetism, ed. L. Que, Jr, University Science Books, Sausalito, CA, 2000. 

ESEEM is a pulsed EPR technique which is complementary to both conventional EPR and ENDOR spectroscopy(74.75). In the ESEEM experiment, one selects a field (effective g value) in the EPR spectrum and through a sequence of microwave pulses generates a spin echo whose intensity is monitored as a function of the delay time between the pulses. This resulting echo envelope decay pattern is amplitude modulated due to the magnetic interaction of nuclear spins that are coupled to the electron spin. Cosine Fourier transformation of this envelope yields an ENDOR-like spectrum from which nuclear hyperfine and quadrupole splittings can be determined. 

Y. Deligiannakis, M. Louloudi, and N. Hadjiliadis, Coord. Chem. Rev. 2000, 201, 1. A thorough review of the application of ESEEM spectroscopy to problems in inorganic chemistry prior to 2000. This is an excellent article for someone new to the field and a fine reference for those who use ESEEM and ENDOR in their research. 

A large variety of hyperfine spectroscopy methods exist that allow the detection of hyperfine and nuclear quadrupole interactions electron spin-echo envelope modulation (ESEEM), ENDOR, and ELDOR-detected NMR (electron-electron doubleresonance detected nuclear magnetic resonance) [13]. Although there are cases in which ESEEM and ENDOR perform equally well, ESEEM-like methods tend to be... 

Advanced EPR techniques such as CW and pulsed ENDOR, electron spin-echo envelope modulation (ESEEM), and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) have been successfully used to examine complexation and electron transfer between carotenoids and the surrounding media in which the carotenoid is located. 

Valuable spectroscopic studies on the dithiolene chelated to Mo in various enzymes have been enhanced by the knowledge of the structure from X-ray diffraction. Plagued by interference of prosthetic groups—heme, flavin, iron-sulfur clusters—the majority of information has been gleaned from the DMSO reductase system. The spectroscopic tools of X-ray absorption spectroscopy (XAS), electronic ultraviolet/visible (UV/vis) spectroscopy, resonance Raman (RR), MCD, and various electron paramagnetic resonance techniques [EPR, electron spin echo envelope modulation (ESEEM), and electron nuclear double resonance (ENDOR)] have been particularly effective probes of the metal site. Of these, only MCD and RR have detected features attributable to the dithiolene unit. Selected results from a variety of studies are presented below, chosen because their focus is the Mo-dithiolene unit and organized according to method rather than to enzyme or type of active site. 

ESEEM spectroscopy is a time-domain (i.e. pulsed) analog of EPR see Electron Spin Echo Envelope Modulation Spectroscopy). In principle, ESEEM contains the same information as is found in EPR and ENDOR, although in practice ESEEM is much more sensitive to weakly coupled nuclei that are not easily detected by ENDOR. On the other hand, strongly coupled nuclei can be undetectable by ESEEM, thus the combination of both techniques is often useful. 

None directly the MHQ technique yields a frozen powder, which can be analyzed by various types of low-temperature spectroscopy like X-, Q-band EPR, UV-Visible spectroscopy, resonance Raman and potentially or in the near future by MCD, Mossbauer, ESEEM, ENDOR, EXAFS, W-, D-band EPR, MAS-NMR and FTIR spectroscopy. 

Comparison of EPR [25] and ESEEM [26] spectra of the NiFe-hydrogenases in HjO and D2O indicates the presence of exchangable protons in the vicinity of the nickel in the Ni-C state. Q-band ENDOR spectroscopy [27] indicates two types of exchangable protons. These exchangable protons only interact weakly with nickel and so it is concluded that they are outside the first coordination... 

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. ... 

Fig. 6 Pulse-Sequences, different pulse sequences for hyperfine and dipolar spectroscopy (ESEEM, ENDOR, and PELDOR)...

Hyperfine spectroscopy methods, such as ID ESEEM, 2D HYSCORE, and ENDOR, have been employed to determine the coordination sphere of Mn " ions bound to the HHRz or to the Diels-Alder ribozyme. The HHRz was first investigated by X-band stimulated echo ESEEM by Britt and coworkers [102]. The ESEEM data revealed nitrogen hyperfine coupling of 2.3 MHz,... 

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. 

---