Frequency-domain ESEEM spectra

Fourier transformation of the modulated time-domain signal produces a frequency-domain ESEEM spectrum. For matrix nuclei, the ESEEM spectrum contains a narrow single line centered at the Larmor precession frequency of the nuclei in the magnetic field of the spectrometer. Thus the position of ESEEM lines in the frequency spectrum allows direct... 

Figure 1 Two-pulse or primary ESEEM data collected for the type-1 Cu(II) site of the Fet3p enzyme. Figure 1(a) shows the time domain data recorded under the following conditions microwave frequency, 9.6883 GHz field strength, 337.0 mT pulse power, 250 W 90° pulse length, 16 ns full width at half maximum (FWHM) sample temperature, 10 K. Figure 1(b) shows the ESEEM spectrum derived from the data of Figure 1(a) by subtraction of a biexponential decay function, application of a Hamming window function, and Fast Fourier Transformation (FFT). The absolute value spectrum is displayed...

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. 

The frequencies and the amplitude of the modulation measured in an ESEEM experiment can both be simulated to derive information about the hyperfine interaction. The ESEEM-derived spectrum in the frequency domain is related to, but not necessarily identical to, an ENDOR spectrum. Thorough reviews of this technique and its analysis are found elsewhere" here we give a brief comparison between the information derived from ESEEM and that directly determined by ENDOR. In an ESEEM experi-... 

In principle, the technique can also be extended to the fitting of pulsed EPR spectra using the frequency domain approach [62]. Each peak in the ESEEM spectrum is given by one or more frequencies, a complex intensity, and a linewidth. Derivatives of the ESEEM frequencies with respect to each of the spin Hamiltonian parameters can be calculated and utilized in an identical fashion to that used for CW spectra simulations. 

Figure 10. Top Normalized time-domam ESEEM spectra of (a) VO(pic)2 in rat liver (b) VOSO4 in rat liver (c) resultant spectrum after subtraction of 0.633(b) firom (a). Bottom Frequency-domain difference ESEEM spectra of VO(pic)2 in kidney (d) and liver (e) after subtraction of amine nitrogen signal (1) ESEEM spectrum of VO(pic)2 in ethylenegly-cokwater (1 1) glass. Reprinted with permission from [60]. Copyright 1995, Elsevier.

Medina and Cammack also performed three-pulse ESEEM on this species a prominent hfc thought to be due to N1 or N3 was detected. They were also able to observe small changes to the frequency-domain spectrum when substrate was bound to the enzyme. 

Medina and Cammack also performed three-pulse ESEEM on this radical, but the frequency-domain spectrum obtained was not considered very informative, although a strong transition was observed which the authors assigned to either N1 or N3. ... 

Muns ENDOR mvolves observation of the stimulated echo intensity as a fimction of the frequency of an RE Ti-pulse applied between tlie second and third MW pulse. In contrast to the Davies ENDOR experiment, the Mims-ENDOR sequence does not require selective MW pulses. For a detailed description of the polarization transfer in a Mims-type experiment the reader is referred to the literature [43]. Just as with three-pulse ESEEM, blind spots can occur in ENDOR spectra measured using Muns method. To avoid the possibility of missing lines it is therefore essential to repeat the experiment with different values of the pulse spacing Detection of the echo intensity as a fimction of the RE frequency and x yields a real two-dimensional experiment. An FT of the x-domain will yield cross-peaks in the 2D-FT-ENDOR spectrum which correlate different ENDOR transitions belonging to the same nucleus. One advantage of Mims ENDOR over Davies ENDOR is its larger echo intensity because more spins due to the nonselective excitation are involved in the fomiation of the echo. 

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