Two-dimensional ESEEM

Dikanov, S. A. and M. K. Bowman (1995). Cross-peak lineshape of two-dimensional ESEEM spectra in disordered S = l/2,1=1/2 spin systems. J. Magn. Reson. A 116 125-128. 

The microwave bridge is stable for days making two-dimensional ESEEM experiments routine. 

Abstract Multi-resonance involves ENDOR, TRIPLE and ELDOR in continuous-wave (CW) and pulsed modes. ENDOR is mainly used to increase the spectral resolution of weak hyperfine couplings (hfc). TRIPLE provides a method to determine the signs of the hfc. The ELDOR method uses two microwave (MW) frequencies to obtain distances between specific spin-labeled sites in pulsed experiments, PELDOR or DEER. The electron-spin-echo (ESE) technique involves radiation with two or more MW pulses. The electron-spin-echo-envelope-modulation (ESEEM) method is particularly used to resolve weak anisotropic hfc in disordered solids. HYSCORE (Hyperfine Sublevel Correlation Spectroscopy) is the most common two-dimensional ESEEM method to measure weak hfc after Fourier transformation of the echo decay signal. The ESEEM and HYSCORE methods are not applicable to liquid samples, in which case the FID (free induction decay) method finds some use. Pulsed ESR is also used to measure magnetic relaxation in a more direct way than with CW ESR. 

Two-dimensional ESEEM (HYSCORE) of Rieske-type Fe-S Clusters... 

Dikanov SA, Xun L, Karpiel AB, Tyryshkin AM, Bowman MK. 1996. QrientationaUy-selected two-dimensional ESEEM spectroscopy of the Rieske-t5rpe iron-sulfur cluster in 2,4,5-trichlorophenoxyacetate monooxygenase from Burkholderia cepacia ACllOO. J Am Chem Soc 118 8408-8416. 

Dikanov SA, Davydov RM, Graslund A, Bowman MK. 1998. Two-dimensional ESEEM spectroscopy of nitrogen h3q)erfine couplings in methemerythrin and azi-domethemerythrin. J Am Chem Soc 120 6797-6805. 

This report was followed by several studies that used pulsed EPR spectroscopy to determine the fete of chelated vanadyl complexes in the organs of chronically treated rats. Fukui et al. measured the 2- and 3-pulse ESEEM spectra of rat kidney, liver, and bone from animals treated with VOSO4 or VO(pic)2 [71]. Dikanov and coworkers used ESEEM and the two-dimensional ESEEM technique hyperfine sublevel correlation (HYSCORE) spectroscopy to examine in detail the coordina-... 

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. 

Advanced EPR techniques such as CW and pulsed ENDOR, electron spin-echo envelope modulation (ESEEM), and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) have been successfully used to examine complexation and electron transfer between carotenoids and the surrounding media in which the carotenoid is located. 

In a paper that appeared in 1979, R.P.J. Merks and R. DeBeer pointed out that the sinusoidal dependence of the stimulated echo ESEEM experiment on x and T (equation 8), presented the opportunity to collect ESEEM data in both time dimensions and then apply a two-dimensional EFT to derive two important benefits. The first benefit was that suppression-free spectra should be obtained along the zero-frequency axis for each dimension while the second benefit would be the appearance of cross-peaks at (tUo, cofs) and (tw, co ) that would allow one to identify peaks that belonged to the same hyperfine interaction. This ESEEM version of the NMR COSY experiment (see Nuclear Magnetic Resonance (NMR) Spectroscopy of Metallobiomolecules) would prove invaluable for ESEEM analysis of complex spin systems. However, the disparity in spin relaxation times in the x and T time dimensions precluded the general application of this method. 

Overlap of lines can make analysis difficult when several nuclei contribute in the one-dimensional (ID) two- and three-pulse ESEEM spectra. Eollowing the development in NMR, methods to simplify the analysis involving two-dimensional (2D) techniques have therefore been designed. The Hyperfine Sublevel Correlation Spectroscopy, or HYSCORE method proposed in 1986 [14] is at present the most commonly used 2D ESEEM technique. The HYSCORE experiment has been applied successfully to study single crystals, but is more often applied to orienta-tionally disordered systems. It is a four-pulse experiment (Fig. 2.23(a)) with a k pulse inserted between the second and the third k/2 pulse of the three-pulse stimulated echo sequence. This causes a mixing of the signals due to the two nuclear transitions with m.s = Vi of an 5 = Vi species. For a particular nucleus two lines appear at (v , V ) and (V ", v ) in the 2D spectrum as shown most clearly in the contour map (d) of Fig. 2.23. The lines of a nucleus with a nuclear Zeeman frequency... 

As mentioned in Chapter 2 overlap of lines can make analysis difficult when several nuclei contribute in the one-dimensional (ID) ESEEM spectra. The HYSCORE method is at present the most commonly used two-dimensional (2D) ESEEM technique to simplify the analysis. Contour maps obtained after 2D Eourier transformation of the echo decay signal followed by projection on the frequency plane are mainly employed for visual or computer analysis to obtain the anisotropic hyperfine couplings. Software for the data processing to obtain the contour is often provided with commercial instruments. Tools for ID and 2D Eourier transforms are also available in commercial software like Matlab. 

Separation of interactions allows for precise measurements of the small interactions of the observed electron spin with remote spins in the presence of line broadening due to larger contributions. Such techniques are therefore most useful for solid materials or soft matter, where ESR spectra are usually poorly resolved. The most selective techniques for isolating one type of interaction from all the others are pulsed double resonance experiments, such as ENDOR and electron-electron double resonance (ELDOR), which are discussed in more detail in Chapter 2. If the hyper-fine couplings are of the same order of magnitude as the nuclear Zeeman frequency, ESEEM techniques may provide higher sensitivity than ENDOR techniques. In particular, the two-dimensional hyperfme sublevel correlation (HYSCORE) experiment provides additional information that aids in the assignment of ESEEM spectra. These experiments are also discussed in Chapter 2. 

Figure 1. Scheme of the pulse EPR sequences mentioned in this chapter, (a) Two-pulse ESEEM. (b) Three-pulse ESEEM. (c) Four-pulse ESEEM. When times fi and ti are stepped under the constraint of ti= ti= T, combination-peak experiment is performed. Two-dimensional HYSCORE spectroscopy is done using the same sequence, whereby t and are stepped independently. The second and third nil pulse are replaced by high-tuming-angle (HTA) pulses in a matched HYSCORE experiment, (d) SMART-HYSCORE. The first and third pulses are HTA pulses, (e) Davies ENDOR. (f) Mims ENDOR. (g) ELDOR-detected NMR. 

Buy C, Matsui T, Andrianambinintsoa S, Sigalat C, Girault G, Zimmerman J-L. 1996. Binding sites for Mg(II) in IT -ATPase from Bacillus PS3 and in the a3b3g subcomplex studied by one-dimensional ESEEM and two-dimensional HYSCORE spectroscopy of oxovanadium(IV) complexes a possible role for b-His-324. Biochemistry 35 14281-... 

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