EPR/ENDOR spectroscopy

Keywords Radiation Damage to DNA, EPR/ENDOR Spectroscopy, Primary Radiation Induced... 

This review has spanned many years of work devoted to the attempts to understand the effects of radiation damage to DNA. The emphasis has been on the use of EPR/ENDOR spectroscopy to reveal the structures of the primary radiation induced products in DNA. ENDOR was invented before 1960, but it took quite some time before this technique was used to study problems in radiation biology. The basic reason is that complex equipment had to be designed and tested that permits the irradiation and examination of small single crystals at helium temperatures. The apparatus was only completed around 1975 by Bernhard and co-workers in Rochester, and by Huttermann and co-workers in Regensburg. 

Early EPR work on sugar radicals led to some questionable radical assignments. There is a need to repeat some of these studies with EPR/ENDOR spectroscopy. It is therefore very encouraging to see new papers in this field with titles like Q-band EPR and ENDOR of Low Temperature X-Irradiated (3-D-Fructose Single Crystals [114] which is using all of the techniques described here to great advantage. 

Carl PJ, Vaughan DEW, Goldfarb D (2002) Interactions of Cu(II) ions with framework Al in high Si Al zeolite Y as determined from X- and W-band pulsed EPR/ENDOR spectroscopies. J Phys Chem B 106 5428-5437... 

Davydov R, Osborne RL, Shanmugam M, Du J, Dawson JH, Hoffman BM (2010) Probing the oxyferrous and catalytically active ferryl states of Am-phitrite omata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I. J Am Chem Soc 132 14995-15004... 

Davydov R, Perera R, Jin SX, Yang TC, Bryson TA, Sono M, Dawson JH, HofFman BM. 2005. Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction EPR/ENDOR spectroscopy. J Am Chem Soc 127(5) 1403-1413. 

Tamanini E, Rigby SEJ, Motevalli M, Todd M, Watkisnon M (2009) Responsive metal complexes a click-based allosteric scorpionate complex permits the detection of a biological recognition event by EPR/ENDOR spectroscopy. Chem Eur J 15 3720-3728... 

Bar, G., M. Bennati et al. (2001). High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy The tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast. J. Am. Chem. Soc. 123 3569-3576. 

EPR, ENDOR, Mossbauer, EXAFS, and MCD spectroscopies to further elucidate intimate details of electron and proton transfer within nitrogenase are difficult... 

The inactive form GOin, which displays a typical Cu(II) EPR signal, yields upon one-electron oxidation the EPR silent active form GO0X. For many years the presence of a Cu(III) ion (ct,. S = 0) in the active site (121) of the fully oxidized state GO0X was assumed. The Whittakers (122) showed in 1990 that one-electron oxidation of the copper depleted apoenzyme of GO produced an EPR active, remarkably stable Tyr radical that was studied by UV-vis, EPR, and ENDOR spectroscopy. From these studies, they concluded that the thioether modified Tyr 272 was oxidized and, consequently, they proposed that GOcx contains a Tyr 272 radical coordinated to a Cu(II) ion. 

The phenomenon of asymmetric hfs tensors was first discussed by McConnell134). Later, Kneubiihl135,136) proved the existence of asymmetric g and A tensors in paramagnetic systems with low symmetry. Evaluation of the asymmetry of A using EPR and ENDOR spectroscopy has been treated by several authors132,137 141). Recently, low-symmetry effects in EPR have been covered in a comprehensive review article by Pilbrow and Lowrey142). 

This section covers the literature of ENDOR on transition metal complexes with organic ligands through the end of 1980. The discussion also includes unpublished results and papers submitted for publication which came to our knowledge. Only contributions for which ENDOR spectroscopy yields either new or significantly improved results compared with the data obtained from EPR measurements will be discussed in more detail. 

Cu(II) impurity complexes in amino acid single crystals have been the subject of several EPR studies181-183. Since nitrogen and proton hf structures are only partially resolved in the EPR spectra, no detailed information about the electronic properties of the complex in the neighborhood of the metal ion can be evaluated. ENDOR spectroscopy has therefore been applied58,63 to draw detailed pictures of the positions and the molecular environment of Cu(II) impurities in amino acid crystals. 

To evaluate the full proton hfs tensors for the rigid molecule and to study the temperature dependence of the dynamics of the ring rotation, ENDOR and EI-EPR spectroscopy has been applied to powder samples of these two systems37,78). EPR, ENDOR and EI-EPR data of V(bz)2 diluted into Fe(cp)2 are summarized in Table 18. 

To resolve hf and nuclear quadrupole interactions which are not accessible in the EPR spectra, George Feher introduced in 1956 a double resonance technique, in which the spin system is simultaneously irradiated by a microwave (MW) and a radio frequency (rf) field3. This electron nuclear double resonance (ENDOR) spectroscopy has widely been applied in physics, chemistry and biology during the last 25 years. Several monographs2,4 and review articles7 11 dealing with experimental and theoretical aspects of ENDOR have been published. 

During the last few years the versatility of ENDOR spectroscopy has been improved by a number of new techniques which make use either of special types of pumping fields (CP-ENDOR, PM-ENDOR), of more than one rf field (DOUBLE ENDOR, multiple quantum transitions, nuclear spin decoupling) or a different display of the spectrum (EI-EPR). In addition to these techniques, alternative methods have been developed (electron spin echo and electron spin echo ENDOR) which are able to supplement or to replace the ENDOR experiment under certain conditions. The utility of all these various advanced techniques, particularly in studies of transition metal compounds, has recently been demonstrated. 

As will be explained in Chapter 7, spectroscopic methods are a powerful way to probe the active sites of the hydrogenases. Often spectroscopic methods are greatly enhanced by judicious enrichment of the active sites with a stable isotope. For example, Mossbauer spectroscopy detects only the isotope Fe, which is present at only 2.2 per cent abundance in natural iron. Hydrogen atoms, which cannot be seen by X-ray diffraction for example, can be studied by EPR and ENDOR spectroscopy, which exploit the hyperfine interactions between the unpaired electron spin and nuclear spins. More detailed information has been derived from hyperfine interactions with nuclei such as Ni and Se, in the active sites. In FTTR spec-... 

Electron-nuclear double resonance (ENDOR) spectroscopy A magnetic resonance spectroscopic technique for the determination of hyperfine interactions between electrons and nuclear spins. There are two principal techniques. In continuous-wave ENDOR the intensity of an electron paramagnetic resonance signal, partially saturated with microwave power, is measured as radio frequency is applied. In pulsed ENDOR the radio frequency is applied as pulses and the EPR signal is detected as a spin-echo. In each case an enhancement of the EPR signal is observed when the radiofrequency is in resonance with the coupled nuclei. 

Once the oxy complex is formed, a second electron transfer to the HO heme effectively reduces the oxy complex to the peroxide level. From this point many heme enzymes catalyze the heterolytic fission of the peroxide 0-0 bond, leaving behind the well known oxyferryl center, (Fe-0) +, characteristic of peroxidase compound 1 and similar to the active hydroxylating intermediate thought to operate in P450s. However, in HO the active oxidizing intermediate is peroxide. Peracids that form the (Fe-0) + intermediate do not support the HO reaction, whereas H2O2 addition to Fe + HO does support substrate hydroxylation 187, 188). EPR and ENDOR spectroscopy have been used to analyze the cryo-genically reduced oxy-HO complex 189). In these studies reduction of... 

In the absence of a 3-dimensional crystal strodure a rathd detailed picture of the active site of aconitase has been generated by the application of a variety of qrectro-scopic techniques. Most important of these has been the melding of informadmi from EPR, Mdssbauer and ENDOR spectroscopy. The future determination of the... 

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. 

In the absence of suitable crystals for an X-ray structural study, combinations of FTIR, RR, XAS, EPR, ENDOR, XANES, and ESEEM spectroscopies were used to characterize the manganese site in the OEC. Vibrational spectroscopy, particularly low-frequency FT infrared and... 

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