ENDOR (Electron-Nuclear DOuble Resonance) spectroscopy is an EMR technique that often is described as EPR-detected NMR. It is very useful for learning about the structure of paramagnetic molecules, as well as information about the distances and orientations of atoms surrounding paramagnetic centers (e.g. proteins and enzymes containing paramagnetic ions, and defects in solids). ENDOR makes use of the electron-nuclear hyperfine couplings (I . A . S) between the unpaired electrons and neighboring nuclei with I != 0, and measures them with much higher precision than may be possible by EPR spectroscopy alone. Because it examines nuclei in the vicinity of unpaired electrons, ENDOR can provide information that conventional NMR misses, due to the short relaxation times of these nuclei. The technique uses two frequencies: a fixed microwave frequency to partially saturate electronic Zeeman transitions and monitor the intensity of the EPR signal, and a strong radiofrequency which is varied in order to excite nuclear (NMR) Zeeman transitions. At a magnetic field (Bo) of 0.34 T, the microwave frequency for a g=2 paramagnetic species would be about 9.5 GHz, while radiofrequencies suitable for driving proton NMR transitions would center around 14 MHz. By drivin g NMR transitions with the strong rf field, a particular nuclear transition rate is enhanced. This is communicated to the electrons by means of the hyperfine coupling between that nucleus and the electron, increasing its induced relaxation rate, which changes the EPR signal intensity of these partially saturated electronic transitions. In this way, ENDOR reports on the NMR of nuclei in the vicinity of paramagnetic electrons.
ENDOR spectroscopy usually is performed on paramagnetic species in liquids and solids. In liquids, ENDOR spectra typically give information on the isotropic hyperfine couplings, while in solids (single crystals, powders, frozen solutions) both isotropic and anisotropic hyperfine interactions are observed. The analysis of ENDOR spectra requires a careful computer simulation in order to determine the precise values of the hyperfine interactions. This information, which may take the form of a matrix for each nucleus, can, in turn, be analyzed to determine the distances and orientations of I != 0 nuclei in the vicinity of the paramagnetic electrons. In solid systems with electronic Zeeman anisotropy, additional information is available from the Bo dependence of the ENDOR spectra, a phenomenon known as "orientation selection." The ability to perform ENDOR at several different magnetic fields is very useful in studying systems of this type (which include most proteins and enzymes that contain transition metal ions, for example), as well as for characterizing the effects of motion on ENDOR spectra. Because the rates of electronic and nuclear transitions must be made comparable in order to observe a strong ENDOR effect, the technique also requires precise temperature control, often at very low (liquid helium) temperatures.
The IERC has experience performing ENDOR on paramagnetic molecules [1,2], as well as on frozen solutions of proteins [3], to mention two examples from recent studies. We have a highly modified commercial ENDOR spectrometer operating at a microwave frequency of 9.5 GHz (X-band), complete with variable temperature capabilities down to 4.2 K. A prototype system operating at 35 GHz (Q-band) is also available for collaborative work. Future plans call for the construction of an ENDOR facility at 95 GHz (W-band). The Center has developed computer simulation techniques for the analysis of ENDOR spectra from disordered solids, including spectra exhibiting "orientation selection," as well as for S >= 1/2 systems. While all current ENDOR spectrometers operate in a cw mode, the IERC also is developing pulsed ENDOR at several frequencies, and these capabilities should be available in the next several years.
1. "ENDOR of Perylene Radicals Adsorbed on Alumina and Silica-Alumina Powders. I. The Ring Protons", R. B. Clarkson, R. L. Belford, K. Rothenberger, and H. Crookham, J. Catalysis, 106, 500 (1987).
2. "ENDOR of Perylene Radicals Adsorbed on Alumina and Silica-Alumina Powders, II. Matrix ENDOR", K. Rothenberger, H. Crookham, R. B. Clarkson, R. L. Belford, J. Catalysis, 115, 430 (1989).
3. "Nitrogen and Proton ENDOR of Cytochrome d, Hemin, and Metmyoglobin in Frozen Solutions", F. Jiang, T. M. Zuberi, J. B. Cornelius, R. B. Clarkson, R. B. Gennis, and R. L. Belford, J. Am. Chem. Soc., 115, 10293-10299 (1993).
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