We have recently implemented a 140 GHz pulsed ENDOR spectrometer for detailed studies of the hyperfine structure and the electron spin density of different nuclei, specifically 13C, 2H and 1H. The spectra obtained are the highest frequency pulsed ENDOR spectra currently available. The high resolution of this technique allows for the complete separation of hyperfine spectra of different nuclei, in contrast to conventional ENDOR at low frequencies. For instance, the 2H ENDOR spectrum of deuterated BDPA (bisdiphenylene phenyl allyl) is almost not resolvable with 9 GHz ENDOR due to the low 2H-Larmor frequency (2.3 MHz) and overlap with 13C-resonances, whereas two distinct doublets are resolved at 140 GHz. The peaks arise from the hyperfine splittings of two inequivalent sets of deuterons. ?Different types of pulsed ENDOR excitation and detection schemes (Davies-, Mims-, TRIPLE-, Refocused ENDOR) have been used in order to demonstrate the sensitivity of the spectrometer. The performance of different pulse sequences is strongly influenced by the application of long soft pulses. The largest ENDOR effect was observed in Mims-ENDOR; however, the spectral line shape suffers from typical blindspots at A = n/t, where A is the hyperfine constant and t is the time between the two 900 excitation pulses. Blindspots can mostly be removed by use of the Refocused-Mims technique. With our current ENDOR spectrometer, we obtained the best sensitivity employing Davies-ENDOR with FID detection. A long, soft 1800 preparation pulse provides for high selectivity in the excitation of the EPR line and produces a narrow hole (< 1 MHz) in the ENDOR spectrum, leading to high sensitivity for small hyperfine couplings. The FID signal can be detected and integrated with minor error without phase cycling, due to the intrinsic short dead time of the 140 GHz spectrometer (< SO ns). The S/N of the FID detected spectra is a factor 10 higher than obtained with conventional spin-echo detection. The substantial g-anisotropy of most organic radicals at S T allows orientation selectivity in the ENDOR experiment. The greater resolution and sensitivity at high fields has been exploited to obtain Davies-ENDOR spectra of the tyrosyl radical in RNR-B2 from E. coli. ENDOR spectra were recorded at different field positions within the rhombic EPR powder pattern. In the small coupling region (A < 5 MHz) we observe resonances from the 2,6-ring- and the weakly coupled ~3-methylene protons. The structure of the spectra is complicated due to the overlapping of different lines, but a pair of doublets is resolved for excitation on the edges of the EPR spectrum (gz and gx), where only few orientations contribute to the ENDOR spectrum. A simulation program was developed which is capable of calculating the ENDOR spectrum for different preparation and detection schemes. The simulations reproduce the essential features of the experimental spectra. We obtained a set of hyperfine couplings in excellent agreement with the parameter set proposed by Hoganson et al.
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