HYSCORE

JEFFREY HARMER, GEORGE MITRIKAS, and ARTHUR SCHWEIGER 

Since time t is varied and ijo and 20 are kept constant, the echo is modulated with the frequency ro = ro - rop, which, for the weak coupling case, becomes cy = [4sl- Therefore, despite some peculiarities [25], this experiment allows for the measurement of undistorted hyperfine spectra. 

The ID methods described above result in undistorted ESEEM spectra and thus can drastically improve resolution. However, in multinuclear spin systems having strongly coupled nuclei with small gyromagnetic ratios and weakly coupled nuclei with large gyromagnetic ratios, peaks may overlap and die spectrum can be complicated and difficult to analyze. The resolution can be further increased by implementing the HYSCORE experiment where times /] and 2 are ineremented independently [28]. As a consequence of the transfer of nuclear eoherence by the n pulse, this 2D experiment correlates nuclear frequencies from different manifolds. For an 5 = A, I = A spin system and ideal pulses the modulation formula for the HYSCORE experiment can be written as [29] 

The advantage of Acjmax for inferring the anisotropic part of the hyperfine interaction arises from the fact that the intensities of the endpoints of the arcs (corresponding to the principal values) vanish since here the depth parameter A = 0. Therefore, the hyperfine parameters cannot be determined easily from the extension of the ridges perpendicular to the diagonal. In addition to this approach, the lineshapes of ESEEM spectra for 5 = 54, / = 54 spin systems have been thoroughly studied [34], and useful representations of the correlation patterns in order to determine ajso and T have been proposed [35]. 

---

Figure Bl.15.12. ESEEM spectroscopy. (A) Top energy level diagram and the corresponding stick spectrum for the two allowed (a) and two forbidden (f) transitions. Bottom time behaviour of the magnetization of an allowed (a) spin packet and a forbidden (f) spin packet during a two-pulse ESE sequence (see figure Bl.15.11 (A)). (B) The HYSCORE pulse sequence.

An alternative 2D ESEEM experiment based on the four-pulse sequence depicted in figure B 1.15.12(B) has been proposed by Meliring and coworkers [40]. In the hyperfme sublevel correlation (HYSCORE) experiment, the decay of the echo intensity as a fimction of is governed by -relaxation, whereas the echo decay along the t2-axis is... 

Hdfer P, Grupp A, Nebenfuhr H and Mehring M 1986 Hyperfine sublevel correlation (HYSCORE) spectroscopy a 2D ESR investigation of the squaric acid radical Chem. Phys. Lett. 132 279-82... 

Use of CW ENDOR techniques to detect P-proton hyperfine couplings and matrix nuclei Pulsed ENDOR techniques to detect P-proton hyperfine couplings and matrix nuclei HYSCORE techniques to detect a-proton anisotropic coupling tensors... 

Density functional theory (DFT) calculations to interpret the powder ENDOR and HYSCORE spectra Establish the use of the g-tensor parameters to detect the presence of dimers... 

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. 

HYSCORE, is a 2D four-pulse ESEEM technique which provides correlation between nuclear frequencies originating from different electron manifolds. The sequence of four microwave pulses is tx/2—x—tx/2—/tx— t2-nl2-x-echo where the echo amplitude is measured as a function of tx and t2 at fixed x. The a-proton anisotropic couplings can be detected by this technique (Konovalova et al. 2001a, Focsan et al. 2008). 

Carotenoid radical formation and stabilization on silica-alumina occurs as a result of the electron transfer between carotenoid molecule and the Al3+ electron acceptor site. Both the three-pulse ESEEM spectrum (Figure 9.3a) and the HYSCORE spectrum (Figure 9.3b) of the canthaxanthin/ A1C13 sample contain a peak at the 27A1 Larmor frequency (3.75 MHz). The existence of electron transfer interactions between Al3+ ions and carotenoids in A1C13 solution can serve as a good model for similar interactions between adsorbed carotenoids and Al3+ Lewis acid sites on silica-alumina. 

FIGURE 9.3 Spectra of the mixture of canthaxanthin (2mM) and A1C13 (2mM) in CH2C12 measured at 60 K at the field B0=3349G and microwave frequency 9.3757 GHz (a) superimposed plot of a set of three-pulse ESEEM spectra as the modulus Fourier transform and (b) HYSCORE spectrum measured with a x=152ns. (From Konovalova, T.A., J. Phys. Chem. B, 105, 8361, 2001. With permission.)... 

D-HYSCORE was used to characterize radicals of zeaxanthin and violaxanthin photo-generated on silica-alumina and to deduce the anisotropic a-proton hyperfine coupling tensors. The couplings (MHz) were assigned based on DFT calculations. From such a comparison, the presence of the neutral radicals formed by proton loss from the radical cations was confirmed. 

The hyperfine coupling tensors of carotenoids were determined from the HYSCORE analysis of the contour line-shapes of the cross-peaks (Dikanov and Bowman 1995,1998, Dikanov et al. 2000), which provided the principal components of the tensors that appear to be rhombic. Such tensors are characteristic of planar conjugated radicals with the unpaired spin in a pz orbital of the carbon of the C-H group. 

HYSCORE spectra of zeaxanthin radicals photo-generated on silica-alumina were taken at two different magnetic fields B0=3450G and B0=3422G, respectively. In order to combine the data from the two spectra, the field correction was applied (Dikanov and Bowman 1998). The correction consists of a set of equations that allow transformation of spectra to a common nuclear Zeeman frequency. The set of new frequencies was added to that of the former spectrum and plotted as the squares of the frequencies v2a and v2p. Examples of these plots can be found in Focsan et al. 2008. 

Carotenoid radical intermediates generated electrochemically, chemically, and photochemically in solutions, on oxide surfaces, and in mesoporous materials have been studied by a variety of advanced EPR techniques such as pulsed EPR, ESEEM, ENDOR, HYSCORE, and a multifrequency high-held EPR combined with EPR spin trapping and DFT calculations. EPR spectroscopy is a powerful tool to characterize carotenoid radicals to resolve -anisotropy (HF-EPR), anisotropic coupling constants due to a-protons (CW, pulsed ENDOR, HYSCORE), to determine distances between carotenoid radical and electron acceptor site (ESEEM, relaxation enhancement). 

HexOMe, methyl glycoside Hex-onic, aldohexonic acid hmba, 2-hydroxy-2-methylbutanoate2 HYSCORE, hyperfine sublevel correlation spectroscopy mod. ampl., modulation amphtude polyGalA, galacturonan Qa, quinic acid XAFS, X-ray absorption fine structure spectroscopy... 

Despite the important role that EPR plays in the characterisation of the surface properties of these transition metal oxides, virtually all of the published papers have continued to utilise traditional cw X-band EPR even though high frequencies and/or additional EPR techniques (such as ENDOR, HYSCORE or ESEEM) could offer enormous advantages. 

Fig. 17. First HYSCORE data from GdDTPA at 4 K, 3502 G. The correlated cross peaks, with splittings from 0.6-1.0 MHz, may reveal 14N-Gd couplings that can be probed [92]...

Silakov, A., Wenk, B., Reijerse, E., and Lubitz, W. (2009). 14N HYSCOR-E investigation of the H-cluster of [FeFe] hydrogenase Evidence for a nitrogen in the dithiol bridge. Phys. Chem. Chem. Phys. 11, 6592—6599. 

Brecht M, van Gastel M, Buhrke T, Friedrich B, Lubitz W. Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy. J Am Chem Soc. 2003 125(43) 13075-83. 

Foerster S, van Gastel M, Brecht M, Lubitz W. An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase. J Biol Inorg Chem. 2005 10(1) 51 62. 

Silakov A, Wenk B, Reijerse E, Albracht SPJ, Lubitz W. Spin distribution of the H-cluster in the Hox-CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans HYSCORE and ENDOR study of 14N and 13C nuclear interactions. J Biol Inorg Chem. 2009 14(2) 301 13. 

---