Electron Nuclear Double Resonance Investigation Research Articles

Tailored Nitroxide Radicals and Biradical Containing 13C Enriched Acetylene Groups: ENDOR and DFT Investigation

A specially synthesized nitroxide biradical R5-C≡13C-(p-C6H4)2-13C≡C-R5 (B4) and two radicals, R5-C≡13CH (RCC) and R5-C≡13C-C6H5 (RCCPh), where R5 is 1-oxyl-2,2,5,5-tetramethyl-pyrroline group, have been studied by X- and W-band electron paramagnetic resonance (EPR) spectroscopy, and by W-band electron-nuclear double resonance (ENDOR). Spin density distribution and hyperfine splitting (hfs) constants on 13C atoms were experimentally determined and also calculated using ORCA 3.0.3 program package. The biradical and radicals geometries were optimized on UKS/B3LYP/cc-pVDZ level. Hfs constants were calculated using density functional theory (DFT) with PBE0 functional and N07D, and were compared with the experimental value of the hfs constant on 13C atoms, measured from ENDOR spectra. It is concluded that at small values of the exchange integral as J ≤ a/2 ≈ 7–8 G, the current quantum chemical approaches do not allow determining precise values of the hfs constants on the 13C atoms in the bridge connecting two paramagnetic nitroxide rings of the biradical.

EPR and 57Fe ENDOR investigation of 2Fe ferredoxins from Aquifex aeolicus

We have employed EPR and a set of recently developed electron nuclear double resonance (ENDOR) spectroscopies to characterize a suite of [2Fe-2S] ferredoxin clusters from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5). Antiferromagnetic coupling between the Fe(II), S = 2, and Fe(III), S = 5/2, sites of the [2Fe-2S](+) cluster in these proteins creates an S = 1/2 ground state. A complete discussion of the spin-Hamiltonian contributions to g includes new symmetry arguments along with references to related FeS model compounds and their symmetry and EPR properties. Complete (57)Fe hyperfine coupling (hfc) tensors for each iron, with respective orientations relative to g, have been determined by the use of "stochastic" continuous wave and/or "random hopped" pulsed ENDOR, with the relative utility of the two approaches being emphasized. The reported hyperfine tensors include absolute signs determined by a modified pulsed ENDOR saturation and recovery (PESTRE) technique, RD-PESTRE-a post-processing protocol of the "raw data" that comprises an ENDOR spectrum. The (57)Fe hyperfine tensor components found by ENDOR are nicely consistent with those previously found by Mössbauer spectroscopy, while accurate tensor orientations are unique to the ENDOR approach. These measurements demonstrate the capabilities of the newly developed methods. The high-precision hfc tensors serve as a benchmark for this class of FeS proteins, while the variation in the (57)Fe hfc tensors as a function of symmetry in these small FeS clusters provides a reference for higher-nuclearity FeS clusters, such as those found in nitrogenase.

An ENDOR and HYSCORE Investigation of a Reaction Intermediate in IspG (GcpE) Catalysis

IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we have specifically assigned the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique fourth Fe in the cluster and C2 and O3 of the ligand.

EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q(B), coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q(B). To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q(B) site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920-6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g(x) = 2.0057, g(y) = 2.0048, g(z) ∼ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.

ENDOR and ESEEM investigation of the Ni-containing superoxide dismutase

Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O2(-) to H(2)O(2) and O(2). The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band (14)N,(1/2)H continuous wave (CW) and pulsed electron-nuclear double resonance (ENDOR) and X-band (14)N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band (14)N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N(delta) of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band (14)N ESEEM characterized the hyperfine coupling of N(epsilon) of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N(epsilon)-H and O(Glu-17) present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band (1)H CW ENDOR and pulsed (2)H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N(delta) or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.

The Mn2+−Bicarbonate Complex in a Frozen Solution Revisited by Pulse W-Band ENDOR

The coordination of bicarbonate to Mn (2+) is the simplest model system for the coordination of Mn (2+) to carboxylate residues in a protein. Recently, the structure of such a complex has been investigated by means of X-band pulse EPR (electron paramagnetic resonance) experiments ( Dasgupta, J. ; et al. J. Phys. Chem. B 2006, 110, 5099 ). Based on the EPR results, together with electrochemical titrations, it has been concluded that the Mn (2+) bicarbonate complex consists of two bicarbonate ligands, one of which is monodentate and other bidentate, but only the latter has been observed by the pulsed EPR techniques. The X-band measurements, however, suffer several drawbacks. (i) The zero-field splitting (ZFS) term of the spin Hamiltonian affects the nuclear frequencies. (ii) There are significant contributions from ENDOR (electron nuclear double resonance) lines of the M S not equal +/- (1)/ 2 manifolds. (iii) There are overlapping signals of (23)Na. All these reduce the uniqueness of the data interpretation. Here we present a high-field ENDOR investigation of Mn (2+)/NaH (13)CO 3 in a water/methanol solution that eliminates the above difficulties. Both Davies and Mims ENDOR measurements were carried out. The spectra show that a couple of slightly inequivalent (13)C nuclei are present, with isotropic and anisotropic hyperfine couplings of A iso1 = 1.2 MHz, T perpendicular1 = 0.7 MHz, A iso2 = 1.0 MHz, T perpendicular2 = 0.6 MHz, respectively. The sign of the hyperfine coupling was determined by variable mixing time (VMT) ENDOR measurements. These rather close hyperfine parameters suggest that there are either two distinct, slightly different, carbonate ligands or that there is some distribution in conformation in only one ligand. The distances extracted from T perpendicular1 and T perpendicular2 are consistent with a monodentate binding mode. The monodentate binding mode and the presence of two ligands were further supported by DFT calculations and (1)H ENDOR measurements. Additionally, (23)Na ENDOR resolved at least two types of (23)Na (+) in the Mn (2+)-bicarbonate complex, thus suggesting that the bicarbonate bridges two positively charged metal ions.

Identification ofCr3+centers inCs2NaAlF6andCs2NaGaF6crystals by EPR and ENDOR paramagnetic resonance techniques

Chromium-doped ${\mathrm{Cs}}_{2}{\mathrm{NaAlF}}_{6}$ and ${\mathrm{Cs}}_{2}{\mathrm{NaGaF}}_{6}$ crystals have been investigated by using the techniques of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) at $X$-band $(9.5\phantom{\rule{0.3em}{0ex}}\mathrm{GHz})$ and $Q$-band $(34\phantom{\rule{0.3em}{0ex}}\mathrm{GHz})$ frequencies. In both crystals, which have the hexagonal elpasolite structure, two inequivalent ${\mathrm{Cr}}^{3+}$ centers are detected. From the angular dependence of the ENDOR spectra for the central $^{53}\mathrm{Cr}$ nucleus and the nearest shells of $^{19}\mathrm{F}$ and $^{23}\mathrm{Na}$ nuclei, the crystallographic sites at which the paramagnetic ${\mathrm{Cr}}^{3+}$ ions are incorporated in these crystals have been unambiguously determined for each type of center. The results of the EPR and ENDOR investigation are discussed in the context of their correlation with optical and crystallographic data for these crystals and give considerable information about local distortions around the paramagnetic impurity ions.

A Pulse EPR and ENDOR Investigation of the Electronic Structure of a σ-Carbon-Bonded Cobalt(IV) Corrole

In this contribution we present a continuous wave (CW), pulse electron paramagnetic resonance (EPR), and pulse electron nuclear double resonance (ENDOR) study of (OEC)Co(C6H5), where OEC is the trianion of 2,3,7,8,12,13,17,18-octaethylcorrole. To facilitate spectral assignments isotopic substitutions were employed (2H and 13C). From the analysis of the frozen solution CW EPR, ESEEM, and ENDOR spectra measured at X- and Q-band, we determined the electronic coupling parameters of the unpaired electron with the cobalt nucleus, corrole nitrogen nuclei, phenyl 13C, 1H and 2H nuclei, meso 1H and 2H nuclei, and ethyl 1H nuclei. Determination of the g matrix alignment in the molecular frame was achieved by successfully simulating the orientationally selective powder ENDOR spectra of the meso nuclei. The g principal values are g1 = 1.9670, g2 = 2.1122, and g3=2.0043, with the g1 and g2 axes pointing at the nitrogens of the corrole macrocycle and the g3 axis directed perpendicular to the plane. The cobalt hyperfine...

A Continuous Wave and Pulse EPR and ENDOR Investigation of Oxygenated Co(II) Corrin Complexes

Heptamethyl cobyrinate perchlorate, [Cob(II)ester]ClO4, has the relevant structural features of base-off B12r, the reduced Co(II) form of vitamin B12. The reversible oxygenation behavior of this complex in different solvents is investigated using continuous wave (cw) EPR at X-band and compared with that of Co(II) porphyrin complexes. Furthermore, the influence of the addition of a nitrogen base (pyridine or 1-methylimidazole) to the solutions is investigated. To determine the electronic structure of the oxygenated complexes, different pulse electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) techniques are applied. The g and cobalt hyperfine matrix and their principal axes are determined using a combination of cw-EPR at X- and Q-band, ESE (electron spin−echo)-detected EPR at W-band and Davies-ENDOR at Q-band. The experimental g and cobalt hyperfine values are found to be sensitive to the change of solvent, addition of a nitrogen base, and change in the ring structure. From ...

ESR, ENDOR, and ESEEM investigations on UV-irradiated 2,4,6-tri-tert-butyl phenol crystals

Electron spin resonance (ESR), electron nuclear double resonance (ENDOR), and electron spin echo envelope modulation (ESEEM) measurements were carried out for UV-irradiated 2,4,6-tri-tert-butyl phenol in the polycrystalline state. The radical produced in the crystal was detected by ESR and identified to be the corresponding phenoxyl radical, which is well characterized in the chemical oxidations in solutions. ENDOR and ESEEM spectra were unambiguously analyzed in terms of the hyperfine coupling constants determined from well-resolved ESR in solutions. Radical pairs in the crystals were also ascertained, and together with the single-crystal study the analysis disclosed zero-field splitting parameters in the triplet states. ESEEM time decays gave relaxation timesT 1 = 5.94 andT 2 = 1.12 μs at room temperature. These appropriate values permit an easy detection of the spin echoes, and therefore this radical matrix can be used as a useful standard for pulsed ESR investigations.

ENDOR Investigation of the Eu2+ Activator in the X-Ray Storage Phosphor BaFBr

Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) have been used to investigate the surrounding of the activator ion Eu 2 in single crystals of the X-ray storage phosphor BaFBr. An analysis of the Eu 2+ superhyperfine interaction with the next fluorine neighbours showed that Eu 2+ substitutes for Ba 2+ and is not shifted from a regular position within experimental error, in spite of the large difference in ionic radii. EPR and ENDOR investigations on BaFBr:Eu 2+ powders established that different methods of producing these materials have no influence on the local environment of Eu 2+ .

Electron paramagnetic resonance and electron nuclear double resonance investigation of NaCl: Ir—a model system for similarly doped silver halides

Iridium-doped Bridgman-grown NaCl single crystals were studied with electron paramagnetic resonance and electron nuclear double resonance after irradiation at 77 K. An orthorhombic-I (IrCl6)4– complex, charge compensated by two next-nearest neighbour vacancies was detected. After annealing at 180 K this complex decays by cither vacancy diffusion or an electronic process. The Ir 2+complex is compared with the Rh2+ complex formed in the same conditions in NaCl crystals grown from solution.

CO Binding to the FeMo Cofactor of CO-Inhibited Nitrogenase: 13CO and 1H Q-Band ENDOR Investigation

The resting state of nitrogenase shows an S = 3/2 electron paramagnetic resonance (EPR) signal resulting from the FeMo-cofactor (MoFe7S9:homocitrate) of the MoFe protein. When the enzyme undergoes turnover under a CO atmosphere, this signal disappears and two new ones appear: one under low pressure of CO (denoted lo-CO; 0.08 atm) and the other under high pressure of CO (denoted hi-CO; 0.5 atm). Our recent Q-band (35 GHz) 13C and 57Fe electron nuclear double resonance (ENDOR) studies demonstrated that one CO is bound to the FeMo-cofactor of lo-CO and two to the cofactor of hi-CO. [Christie, P. D.; Lee, H. I.; Cameron, L. M.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 8707−8709. Pollack, R. C.; Lee, H. I.; Cameron, L. M.; DeRose, V. J.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117, 8686−8687.] In the present report, we examine the CO-bound FeMo-cofactor in both the lo- and hi-CO forms of the MoFe protein from Azotobacter vinelandii by complete orientation-selective 13C and 1H ENDOR measurements. 1H ENDOR studies reveal that well-resolved signals from a solvent-exchangeable proton seen in the resting state FeMo-cofactor are lost in both of the CO-inhibited forms, indicating a loss in hydrogen bonding as compared to the resting state. This supports the hypothesis that CO binds near the “waist” of the cofactor. Determination of 13C hyperfine tensors of bound 13CO to lo-CO and hi-CO leads to the suggestion that the single CO bound to the FeMo-cofactor of lo-CO may bridge or semibridge two iron ions, while each of the two CO bound to hi-CO is a terminal ligand. These ENDOR measurements and recent FTIR results of Thorneley and co-workers [George, S. J.; Ashby, G. A.; Wharton, C. W.; Thorneley, R. N. F. J. Am. Chem. Soc. 1997, 119, 6450−6451] provide strong mutual support.

EPR and ENDOR Investigations of Shallow Impurities in SiC Polytypes

Investigations of nitrogen donors in 6H-, 4H- and 3C-SiC using conventional electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and optical detection of EPR and ENDOR as well as optical absorption and emission spectroscopy are reviewed and critically discussed. An attempt is presented to interpret the experimentally found large differences in hyperfine interactions of the 14N nuclei on the various inequivalent sites in the different polytypes of SiC in terms of valley–orbit splittings and “central-cell corrections” in the framework of the effective mass theory (EMT). P-doping by neutron transmutation in 6H-SiC resulted in various P-related EPR spectra previously associated with shallow P donors and P–vacancy complexes. In analogy to the new interpretation of the N donor spectra in various polytypes, it is proposed that all P-related spectra found hitherto in 6H-SiC are due to isolated P donors in ground and excited EMT states. A detailed discussion is presented of the electronic structure of B acceptors, as determined by EPR and in particular by ENDOR investigations: The B atom itself has only very little unpaired hole density, while the hole resides mainly on a neighbouring relaxed C atom B acceptors have a rather “deep” character and pronounced dynamical properties. A discussion of the present understanding of the so-called deep B centre (D centre) is also given. In contrast to B, the Al acceptor behaves as expected from the effective mass theory. It shows, however, two optical absorption bands identified by optical detection of EPR which are related to an ionization transition to the valence band and another transition, probably to a V impurity.

1H ENDOR investigations on gamma-irradiated betaine calcium chloride dihydrate in the incommensurate phase

The electron nuclear double resonance (ENDOR) technique is applied to study the influence of the incommensurate modulation on the proton hyperfine structure (hfs) tensors of the N(CH3)3 radical in betaine calcium chloride dihydrate (BCCD). The temperature dependence of the edge singularities due to the increasing of the incommensurate (IC) modulation amplitude could be observed in the1H ENDOR of the CH3 groups for the first time. The rotation pattern of the edge singularities observed in the1H spectra in the IC phase of BCCD can be explained by a libration around the C1-N axis with a tilt angle of about ±7°.

EPR and ENDOR investigation of the primary electron acceptor radical anion QA.- in iron-depleted photosystem II membrane fragments.

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

Electron nuclear double resonance of chalcogen donors in GaP revisited

Using electron nuclear double resonance (ENDOR) of GaP crystals containing shallow S donors it was shown that the ENDOR spectra previously detected optically in the two infrared photoluminescence bands assigned to oxygen in oxygen-doped GaP are indeed due to S donors which must have been present simultaneously in those crystals. Furthermore it is shown that the results of the published ENDOR investigations of both S and Te donors in GaP must be revised. Finally the measured ligand hyperfine interaction of S and Te donors are discussed within the frame of effective mass theory.

ENDOR investigation of the microscopic structure of the boron acceptor in 6H-SiC

The boron acceptor in 6H-SiC was investigated using electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR). The hyperfine interactions with 11B could be determined precisely for the two quasi-cubic and the hexagonal sites. The microscopic model suggested from the EPR and ENDOR results is as follows: boron occupies a silicon site and is a negatively charged ligand to an adjacent carbon atom on which most of the unpaired spin density is located. At low temperatures the symmetry of the two quasi-cubic site defects is monoclinic, while the hexagonal site defect has C3V symmetry about the hexagonal axis. At about 50 K the two quasi-cubic site defects experience a thermally activated motion of the hole at the adjacent carbon about the hexagonal crystal axis and the apparent defect symmetry also becomes C3V. The boron acceptor should be regarded as a boron-induced carbon acceptor.

Electron-nuclear double-resonance investigations on Cr3+ ions in natural MgAl2O4 spinel

The ENDOR spectra of Cr3+ ions in natural MgAl2O4 spinel have been measured at 4.2 K and the hyperfine and quadrupole interactions with the nearest-neighbour 27Al nuclei and with the 53Cr isotope have been investigated. The results show that Cr3+ has the point symmetry 3m, being subjected to the original trigonal expansion at the B sites, but inducing some distortion effects on its surroundings. Further, the isotropic hyperfine constant of the 27Al nuclei has been analysed in an independent bonding model, showing that there is appreciable transfer of unpaired spin to the aluminium ion due to covalency.

Electron spin resonance, electron spin echo, and electron nuclear double resonance investigations of the photoreduction yield of a series of alkylmethylviologens in dihexadecyl phosphate vesicles: effect of added cholesterol

The initial event in photosynthesis, photoinduced charge separation, takes place in a membrane bound proton/chlorophyll complex - the photosynthetic reaction center. An electron spin resonance (ESR), electron spin echo (ESE), and electron nuclear double resonance (ENDOR) investigation was performed on a series of alkylmethylviolgens which were solubilized in dihexadecyl phosphate (DHP) vesicles and rapidly frozen at 77 K. Photoreduction yields were measured by ESR, while ESE and matrix ENDOR were used to determine the location of the viologen radical in the lipid bilayer with respect to the water interface and the hydrocarbon interior, respectively. The effect of various concentrations on cholesterol on both the photoreduction yield and the location of the viologen probe within the DHP vesicle was measured. The ESE and ENDOR results indicate that the increased lipophilicity of the longer chained viologens effects deeper solubilization into the bilayer. This increased lipophilicity correlates with an enhancement of the photoreduction yield due to a reduced back electron transfer rate. Addition of cholesterol results in a disruption of the integrity of the vesicle interface and some increase in the photoreduction yield. This increase is mostly probably due to a decrease in effective interface charge density of the vesicle as a resultmore » of the intercalation of cholesterol molecules into the bilayer.« less