EPR and ENDOR

The complete spin Hamiltonian for a description of EPR and ENDOR experiments is given by... 

Almost simultaneously, Lindahl and co-workers proposed that Cluster C is the CO oxidation site based on EPR and ENDOR studies of the cyanide adduct of the enzyme (134). That proposal was based on the premise that CO and cyanide compete for the same binding site. Additionally, Xia and Lindahl have shown that, by mild SDS treatment, they can partially dissociate CODH/ACS, which is a tetra-meric enzyme with an subunit composition, into an isolated a subunit and an form (135). The form has the same level of CO oxidation activity as the native protein indicating that the a subunit is not involved in CO oxidation and that the /8 subunit must contain the clusters required for CO oxidation (135). In addition, CO2 alters the g values of the Credi form of the enzyme (136). 

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. 

Konovalova, T. A. and L. D. Kispert (1998). EPR and ENDOR studies of carotenoid-solid Lewis acid interactions. J. Chem. Soc. Faraday Trans. 94 1465-1468. 

Wu, Y., L. Piekara-Sady et al. (1991). Photochemically generated carotenoid radicals on Nafion film and silica gel An EPR and ENDOR study. Chem. Phys. Lett. 180 573-577. 

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. 

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). 

An EPR and ENDOR investigation of the planar copper complex 63Cu(sal)2 (Fig. 30) substituted into a single crystal of Ni(sal)2 has been reported by Schweiger et al.62,65). The aim of this work was to determine the structure of the internal H-bond occuring in Cu(sal)2, and to draw a detailed picture of the unpaired electron distribution on the... 

An extensive EPR and ENDOR study of Cu(TPP) and Ag(TPP) (Fig. 32) doped into (H20)Zn(TPP) single crystals has been published by Brown and Hoffman66,171. The complete set of hfs and quadrupole tensors for the 14N nuclei and the hfs tensors of the Ag(II) and Cu(II) ions and of the pyrrole protons are reported. A detailed analysis of the magnetic parameters (Table 6.1), which are measured with high accuracy, has been presented using the standard MO treatment66. ... 

Fig. 39a-c. EPR and ENDOR spectra of stellacyanin. a) Experimental EPR spectrum. The fields near which the single crystal-like ENDOR spectra were measured are marked with (a)gz, (b)gy and (c)g temperature 77 K. b) I4N-ENDOR spectrum with B0 along g, temperature 2K. c) Cu-ENDOR spectrum with Bo near position (c) (opposite phase to H- and 14N-ENDOR). (Adapted from Ref. 199)... 

EPR and ENDOR spectra very similar to those found in VO(TPP) have also been observed2885 in a sample of a black solid obtained by chloroform extraction of a phosphate mineral from Youssoufia (Morocco). The close correspondence of the spectra suggests that the black solid contains a vanadyl porphyrin derivative. 

For more complicated spin systems than the one presented in Fig. 1, the number of EPR lines becomes much greater than the number of ENDOR lines. This is due to the different selection rules for EPR and ENDOR transitions. In an EPR spectrum (Ams = 1, Ami = 0) the number of lines increases multiplicatively with the number of nonequivalent nuclei, whereas in ENDOR (Ams = 0, Amt = 1) the corresponding increase is only additive7. Since on the other hand the total spectral widths in EPR and ENDOR are of the same order of magnitude (expressed in units of energy), the average spectral... 

Fig. 2 a, b. EPR and ENDOR spectrum of the low-spin Co(II) Schiff base complex Co(acacen) diluted into a Ni(acacen) 1/2 H20 single crystal, temperature 8K. a) EPR spectrum the two magnetically nonequivalent sites coincide for this particular orientation (EPR observer is marked by an arrow) b) ENDOR spectrum of H, 13C (enriched) and 14N ligand nuclei vp free proton frequency denote the AmN = 2 nitrogen ENDOR transitions. (From Ref. 12)... 

Davydov, R., Makris, T.M., Kofman, V., Werst, D.E., Sligar, S.G. and Hoffman, B. M. (2001) Hydroxylation of camphor by reduced oxy-cytochrome P450cam mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes. Journal of the American Chemical Society, 123, 1403-1415. 

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-... 

Fig. 5. Schematic illustration of the Cu + binding sites in G4-OH and G4-NH2. The models are based on UV-vis, EPR, and ENDOR data...

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... 

Table IV. EPR and ENDOR Results on Aconitase with 1 0 Labeled Substrate,...

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. 

TCP and its metal-substituted derivatives can be made to dimerize quantitatively by the addition of cations (NU, K, Cs, Ba ) to solutions of these porphyrins ( ). The stoichiometry of the dimerization reaction establishes that two porphyrins are linked by four crown-cation-crown bridges ( ). This forces the porphyrins in a parallel configuration. For steric reasons, one ring system must be rotated relative to the other. EPR ( ) and ENDOR (IS) studies of the ground state triplet [CuTCP]2 and [VOTCP12 dimers fit a model of two parallel porphyrins with the transition metal ions positioned on a common axis perpendicular to the planes, with a center-to-center distance of 0.43 nm. 

S. Van Doorslaer and E. Vinck, The strength of EPR and ENDOR techniques in revealing structure-function relationships in metalloproteins, Phys. Chem. Chem. Phys., 9 (2007) 4620-4638. 

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