Electron‐Nuclear Double Resonance ( ENDOR ) Spectroscopy

Abstract Electron‐nuclear double resonance (ENDOR) spectroscopy is a magnetic resonance technique that was invented in the mid 1950s by George Feher, then at Bell Telephone Laboratories. The first applications of ENDOR were to problems in solid state physics, but beginning in the 1970s, and particularly since the late 1980s, ENDOR has been widely applied to bioinorganic systems. ENDOR is fundamentally an electron paramagnetic resonance (EPR) technique, however, it also has aspects of nuclear magnetic resonance (NMR) spectroscopy. Essentially, ENDOR consists of the monitoring of an EPR signal, while effecting NMR transitions. The EPR signal can be either a continuous wave (CW) signal or a pulsed, electron spin echo (ESE) signal, in both cases caused by the application of microwave energy to a sample in an external magnetic field. As with EPR itself, ENDOR can thus be either a CW or pulsed (time‐domain) spectroscopic technique. The NMR transitions are caused by the application of radiofrequency (rf) energy, as with NMR itself. NMR transitions only of nuclei magnetically (hyperfine) coupled to the paramagnetic center (i.e., the center giving rise to the EPR signal) affect the intensity of this EPR signal, whether CW or pulsed, and provide characteristic ENDOR spectra. As a consequence of its double resonance nature, ENDOR has a great deal of flexibility in spectroscopic parameters. The parameters that affect EPR signals, e.g., microwave power, can also play a role in ENDOR spectral appearance. Additionally, there are parameters relevant to the NMR aspects of ENDOR that affect ENDOR spectra. In both CW and pulsed ENDOR there is experimental flexibility, which is described here. As a result, ENDOR spectroscopy can provide detailed information on multiple, magnetically active nuclei that comprise a paramagnetic center in a bioinorganic system. This information includes identification of such nuclei and metrical information on their relation to the paramagnetic center. This article uses a multifaceted case study to illustrate the application of ENDOR spectroscopy in bioinorganic chemistry. Specifically, the case study describes the application of multinuclear ( 1, 2 H, 13 C, 57 Fe, 95 Mo) CW and pulsed ENDOR to enhance our understanding of the iron–molybdenum cofactor in nitrogenase. ENDOR allows identification of the constituent metal ions and their formal oxidation states, and identifies the nature of the substrate analog or inhibitor binding to the cluster. In combination with resting state structural information, and computational and model system studies, ENDOR can provide a structural picture of the intermediates in nitrogen fixation.