Electron nuclear double resonance spectroscopy sensitivity

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. 

ESR-related spectroscopies that hold the potential to overcome some resolution limitations and yield more information than the classical ESR approach about the chemical environment of paramagnetic metal ions are the electron—nuclear double resonance (ENDOR) (Kevan and Kispert, 1976) and electron-spin echo envelope modulation (ESEEM) (Kevan and Schwartz, 1979) spectroscopies. Either ENDOR or ESEEM represents by principle a useful tool in extending resolution of the ESR experiment. However, the sensitivity of ENDOR and ESEEM is much lower than that of ESR, and interpretation of ENDOR and ESEEM spectra is not a simple matter, especially if ligands are not well characterized, as is the case for HSs. Both ENDOR and ESEEM techniques have not yet been applied to strictly metal-HS complexes, but the sensitivity and ease of carrying out experiments are improving rapidly, so major scientific activity may be anticipated to occur in this area of ESR spectroscopy. 

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. 

Electron Spin Echo Envelope Modulation (ESEEM) and pulse Electron Nuclear Double Resonance (ENDOR) experiments are considered to be two cornerstones of pulse EPR spectroscopy. These techniques are typically used to obtain the static spin Hamiltonian parameters of powders, frozen solutions, and single crystals. The development of new methods based on these two effects is mainly driven by the need for higher resolution, and therefore, a more accurate estimation of the magnetic parameters. In this chapter, we describe the inner workings of ESEEM and pulse ENDOR experiments as well as the latest developments aimed at resolution and sensitivity enhancement. The advantages and limitations of these techniques are demonstrated through examples found in the literature, with an emphasis on systems of biological relevance. 

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