Pulsed EPR

23 Electron Paramagnetic Resonance Spectroscopy and Forward Recoil Spectrometry 

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In this article only the most important and frequently applied EPR methods will be introduced. For more extensive treatments of CW and pulsed EPR the reader is referred to some excellent review articles that will be specified in the respective sections of this article. A good starting point for fiirther reading is provided by a number of outstanding textbooks which have been written on the various aspects of EPR in general [2, 3, 4, 5, 6, 7 and 8]. Interested readers... 

As a sununary it may be of interest to point out why TREPR spectroscopy and related methods remain important in the EPR regime, even though pulsed EPR methods are becoming more and more widespread. (1) For the case of an inliomogeneously broadened EPR line the time resolution of TREPR compares favourably with pulsed teclmiques. 

In the previous chapters experiments have been discussed in which one frequency is applied to excite and detect an EPR transition. In multiple resonance experiments two or more radiation fields are used to induce different transitions simultaneously [19, 20, 21, 22 and 23], These experiments represent elaborations of standard CW and pulsed EPR spectroscopy, and are often carried out to complement conventional EPR studies, or to refine the infonnation which can in principle be obtained from them. 

In recent years, however, enomious progress has been made and with the availability of the appropriate MW equipment pulsed EPR has now emerged from its fomier shadowy existence. Fully developed pulse EPR instrumentation is nowadays connnercially available [31, 33]. 

The practical goal for pulsed EPR is to devise and apply pulse sequences in order to isolate pieces of infomiation about a spin system and to measure that infomiation as precisely as possible. To achieve tliis goal it is necessary to understand how the basic instnunentation works and what happens to the spins during the measurement. 

The design of a pulsed EPR spectrometer depends heavily on tlie required pulse lengdi and pulse power which in turn are mainly dictated by the relaxation times of tlie paramagnetic species to be studied, but also by the type of experiment perfomied. When pulses of the order of a few nanoseconds are required (either to compete... 

The electron-spm echo envelope modulation (ESEEM) phenomenon [37, 38] is of primary interest in pulsed EPR of solids, where anisotropic hyperfme and nuclear quadnipole interactions persist. The effect can be observed as modulations of the echo intensity in two-pulse and three-pulse experiments in which x or J is varied. In liquids the modulations are averaged to zero by rapid molecular tumbling. The physical origin of ESEEM can be understood in tenns of the four-level spin energy diagram for the S = I = model system... 

Keljzers C P, Reljerse E and Schmidt J 1989 Pulsed EPR a New Field of Applications (Amsterdam North-Holland)... 

EPR studies of host-guest complexes of carotenoids Measuring distances between carotenoid radicals and distant metals in matrices by using ESEEM methods and pulsed EPR relaxation techniques EPR studies of radical cations on activated alumina and silica-alumina... 

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). 

Focsan, A. L., M. K. Bowman et al. (2008). Pulsed EPR and DFT characterization of radicals produced by photooxidation of zeaxanthin and violaxanthin on silica-alumina. J. Phys. Chem. B 112 1806-1819. 

Recently, due to increased interest in membrane raft domains, extensive attention has been paid to the cholesterol-dependent liquid-ordered phase in the membrane (Subczynski and Kusumi 2003). The pulse EPR spin-labeling DOT method detected two coexisting phases in the DMPC/cholesterol membranes the liquid-ordered and the liquid-disordered domains above the phase-transition temperature (Subczynski et al. 2007b). However, using the same method for DMPC/lutein (zeaxanthin) membranes, only the liquid-ordered-like phase was detected above the phase-transition temperature (Widomska, Wisniewska, and Subczynski, unpublished data). No significant differences were found in the effects of lutein and zeaxanthin on the lateral organization of lipid bilayer membranes. We can conclude that lutein and zeaxanthin—macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity—cannot parallel the ability of cholesterol to induce liquid-ordered-disordered phase separation. 

Kawasaki, K., J.-J. Yin, W. K. Subczynski, J. S. Hyde, and A. Kusumi. 2001. Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane Methodology development and its application to studies of influenza viral membrane. Biophys. J. 80 738-748. 

Subczynski, W. K. and A. Kusumi. 2003. Dynamics of raft molecules in the cell and artificial membranes Approaches by pulse EPR spin labeling and single molecule optical microscopy. Biochim. Biophys. Acta 1610 231-243. 

T. Prisner, M. Rohrer and F. MacMillan, Pulsed EPR spectroscopy Biological applications, Annu. Rev. Phys. Chem., 2001, 52, 279. 

C. Finazzo, J. Harmer, B. Jaun, E.C. Duin, F. Mahlert, R.K. Thauer, S. Van Doorslaer and A. Schweiger, Characterization of the MCRred2 form of methyl-coenzyme M reductase A pulse EPR and ENDOR study, J. Biol. Inorg. Chem., 2003, 8, 586. 

S. Van Doorslaer, R. Bachmann and A. Schweiger, A pulse EPR and ENDOR investigation of the electronic and geometric structure of cobalt-ous tetraphenylporphyrin(pyridine), J. Phys. Chem. A, 1999, 103, 5446. 

Chapman A, Cammack R, Hatchikian EC, et al. 1988. A pulsed EPR study of redox-dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase. FEBS Lett 242 134-8. 

Electron nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) are two of a variety of pulsed EPR techniques that are used to study paramagnetic metal centers in metalloenzymes. The techniques are discussed in Chapter 4 of reference la and will not be discussed in any detail here. The techniques can define electron-nuclear hyperfine interactions too small to be resolved within the natural width of the EPR line. For instance, as a paramagnetic transition metal center in a metalloprotein interacts with magnetic nuclei such as H, H, P, or these... 

ESEEM is a pulsed EPR technique which is complementary to both conventional EPR and ENDOR spectroscopy(74.75). In the ESEEM experiment, one selects a field (effective g value) in the EPR spectrum and through a sequence of microwave pulses generates a spin echo whose intensity is monitored as a function of the delay time between the pulses. This resulting echo envelope decay pattern is amplitude modulated due to the magnetic interaction of nuclear spins that are coupled to the electron spin. Cosine Fourier transformation of this envelope yields an ENDOR-like spectrum from which nuclear hyperfine and quadrupole splittings can be determined. 

Amazingly, low-temperature CW EPR has only been sparsely applied for the study of oxo-Cr(V) complexes,29,52-55 and even fewer studies involve CW ENDOR55,56 or pulsed EPR/ENDOR experiments,52 or attempt a combination of EPR and quantum-chemical computations.29,52,54 A great potential evidently still exists for further research along this line. 

SCHEME 11. Models of oxo-Cr(V) complexes formed with 37 as proposed from room-temperature CW-EPR spectra. It was possible to confirm assignment of only the complexes I and II with low-temperature CW and pulsed EPR/ENDOR and DFT.52... 

Another approach by Hiittermann et al. [50] has been to use pulsed EPR techniques to study the radicals present in DNA fibers equilibrated in D2O and then irradiated and observed at 77 K. This work supports the conclusions that the primary radiation-induced defects are Cyt and Gua. Also reported are contributions from Thy and an allyl radical found on thymine [Thy(Me—H) ]. Also discussed are three components tentatively assigned as adenine and guanine anions and a species whose dominant hyperfine interaction involves the N1 of cytosine. 

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