Spin Echo Envelope Modulation Studies Research Articles

1H-HYSCORE Reveals Structural Details at the Fe(II) Active Site of Taurine:2-Oxoglutarate Dioxygenase.

Proton Hyperfine Sublevel Correlation (1H-HYSCORE) experiments have been used to probe the ligation structure of the Fe(II) active site of taurine:2-oxoglutarate dioxygenase (TauD), a non-heme Fe(II) hydroxylase. To facilitate Electron Paramagnetic Resonance (EPR) experiments, Fe(II) derivatives of the enzyme were studied using nitric oxide as a substitute for molecular oxygen. The addition of NO to the enzyme yields an S = 3/2 {FeNO}7 paramagnetic center characterized by nearly axial EPR spectra with g⊥ = 4 and g|| = 2. Using results from (i) an X-ray crystallographic study of TauD crystallized under anaerobic conditions in the presence of both cosubstrate 2-oxoglutarate and substrate taurine, (ii) a published theoretical description of the {FeNO}7 derivative of this form of the enzyme, and (iii) previous 2H-Electron Spin Echo Envelope Modulation (ESEEM) studies, we were able to assign the proton cross peaks detected in orientation-selected 1H-HYSCORE spectra. Discrete contributions from the protons of two coordinated histidine ligands were resolved. If substrate taurine is absent from the complex, orientation-selective HYSCORE spectra show cross peaks that are less resolved and when combined with information obtained from continuous wave EPR, support an alternate binding scheme for 2-oxoglutarate. HYSCORE studies of TauD in the absence of 2-oxoglutarate show additional 1H cross peaks that can be assigned to two distinct bound water molecules. In addition, 1H and 14N cross peaks that arise from the coordinated histidine side chains show a change in NO coordination for this species. For all of the TauD species, 1H hyperfine couplings and their orientations are sensitive to the detailed electronic structure of the {FeNO}7 center.

Adsorption and Desorption of HD on the Metal–Organic Framework Cu2.97Zn0.03(Btc)2Studied by Three-Pulse ESEEM Spectroscopy

Cu2.97Zn0.03(btc)2 is a structural analogue of the well-known HKUST-1 metal–organic framework. In this compound 1% of the Cu2+ ions in the paddle-wheel units are substituted by Zn2+, resulting in the formation of Cu/Zn paddle-wheel units in low concentration. The paramagnetic Cu2+ ions of these mixed Cu/Zn pairs allow to perform pulsed electron paramagnetic resonance experiments at low temperatures. Here we report on the three-pulse electron spin echo envelope modulation (3p ESEEM) study of the deuterated hydrogen gas HD adsorption and desorption in Cu2.97Zn0.03(btc)2. The HD adsorption sites in this modified compound were identified by precisely simulating experimentally observed 3p ESEEM time domain pattern. To elucidate the HD desorption process, the 3p ESEEM experiments were performed at different temperatures. Employing this method, the detachment of HD from the Cu2+ binding sites is found to already occur slightly above 6 K temperature. Hereby 3p ESEEM spectroscopy reveals to be a powerful method to...

Permethyl-β-Cyclodextrin Spin-Labeled with Nitronyl Nitroxide: Synthesis and EPR Study

A method for synthesis of new covalently linked spin-labeled cyclodextrin (CD) via the attachment of nitronyl nitroxide 2-(4-hydroxyphenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl to permethylated β-cyclodextrin is described. Electron spin echo envelope modulation (ESEEM) studies demonstrate that the new spin-labeled CD exhibits dynamic equilibrium between conformations with radical fragment capping the cavity of CD and radical fragment located outside the cavity. In solution, nitronyl nitroxide attached to CD retains its sensitivity to nitric oxide (NO), as reaction with NO leads to formation of iminonitroxide fragment evidenced by continuous-wave (CW) electron paramagnetic resonance (EPR). At the same time, CW EPR study of the reaction with ascorbic acid shows that the described binding of nitronyl nitroxide to CD does not provide higher stability of radical towards the reduction, and the corresponding rate constants are close to those obtained for free nitronyl nitroxide. Plausible explanations of these observations are discussed.

Hydration Preferences for Mn4Ca Cluster Models of Photosystem II: Location of Potential Substrate–Water Binding Sites

Density functional theory calculations are reported on a set of three model structures of the Mn(4)Ca cluster in the water-oxidizing complex of Photosystem II (PSII), which share the structural formula [CaMn(4)C(9)H(10)N(2)O(16)](q+)·(H(2)O)(n) (q=-1, 0, 1, 2, 3; n=0-7). In these calculations we have explored the preferred hydration sites of the Mn(4)Ca cluster across five overall oxidation states (S(0) to S(4)) and all feasible magnetic-coupling arrangements to identify the most likely substrate-water binding sites. We have also explored charge-compensated structures in which the overall charge on the cluster is maintained at q=0 or +1, which is consistent with the experimental data on sequential proton loss in the real system. The three model structures have skeletal arrangements that are strongly reminiscent, in their relative metal-atom positions, of the 2.9-, 3.7-, and 3.5 Å-resolution crystal structures, respectively, whereas the charge states encompassed in our study correspond to an assignment of (Mn(III))(3)Mn(II) for S(0) and up to (Mn(IV))(3)Mn(III) for S(4). The three models differ principally in terms of the spatial relationship between one Mn (Mn(4)) and a generally robust Mn(3)Ca tetrahedron that contains Mn(1), Mn(2), and Mn(3). Oxidation-state distributions across the four manganese atoms, in most of the explored charge states, are dependent on details of the cluster geometry, on the extent of assumed hydration of the clusters, and in some instances on the imposed magnetic-coupling between adjacent Mn atoms. The strongest water-binding sites are generally those on Mn(4) and Ca. However, one structure type displays a high-affinity binding site between Ca and Mn(3), the S-state-dependent binding-energy pattern of which is most consistent with the substrate water-exchange kinetics observed in functional PSII. This structure type also permits another water molecule to access the cluster in a manner consistent with the substrate-water interaction with the Mn cluster, seen in electron spin-echo envelope modulation (ESEEM) studies of the functional enzyme in the S(0) and S(2) states. It also rationalizes the significant differences in hydrogen-bonding interactions of the substrate water observed in the FTIR measurements of the S(1) and S(2) states. We suggest that these two water-binding sites, which are molecularly close, model the actual substrate-binding sites in the enzyme.

Hydrogen distribution in the vicinity of dangling bonds in hydrogenated amorphous silicon (a‐Si:H)

AbstractWe have investigated the distribution of H atoms around native dangling bonds in a‐Si:H by electron‐nuclear double resonance (ENDOR). In contrast to previous electron spin echo envelope modulation (ESEEM) studies [Isoya et al., Phys. Rev. B 47(12), 7013–7024 (1993)] we find that the distance between H atoms and dangling‐bond defects can be well below r = 3 Å. Our experimental data suggest that the H distribution is continuous and homogeneous and there is no indication for a short‐range order between H atoms and dangling bonds. This work is a first step toward the investigation of the H distribution around light‐induced defects to test models predicting the immediate proximity of H and defects.

Electron spin resonance (ESR) and electron spin echo envelope modulation (ESEEM) studies on the ultramarine analogs obtained from zeolite A with various alkaline cations at different temperatures

Selected pigment samples prepared from mixture of zeolite A, sulfur and relatively low amount of alkaline carbonates (Na, Li, K, and Cs) by thermal treatment at 500 °C or 800 °C were characterized by means of UV–vis, ESR, ED-ESR and ESEEM spectroscopy in order to explain the influence of various cations on their coloration and properties. The samples maintain the original structure of the parent zeolite. The different colors result from the nature of applied cations as well as from the synthesis temperature. It is reflected in UV–vis spectra showing various intensity of the absorption bands assigned to S 2 - and S 3 - anion-radical chromophores. The ESR spectra recorded at 10 K indicated five components attributed to S 3 - . The best quality single-component rhombic spectrum with g x = 2.0011, g y = 2.0510 and g z = 2.0355, appears in the samples prepared with Na and K cations at 800 °C indicating a local homogeneity of the structure around the radicals. The g-factors are analyzed in terms of molecular orbital theory. The other samples (particularly those prepared at 500 °C) are less ordered which is reflected in a strong ESR line broadening along g-factor z-axis. This disorder is explained as due to a distribution of the trisulfur molecule bend angle. The ESEEM spectra of the pigments containing Li and Cs prepared at 500 °C indicate the peaks assigned to 7Li and 133Cs, while for the analogous samples obtained by heating at 800 °C these spectra lack such peaks. This is explained as an effect of a transition from a static disorder to a dynamic disorder visible as a transformation of static ESR spectrum into broad dynamically averaged homogeneous lines.

Structural studies of the molybdenum center of the pathogenic R160Q mutant of human sulfite oxidase by pulsed EPR spectroscopy and 17O and 33S labeling.

Electron paramagnetic resonance (EPR) investigation of the Mo(V) center of the pathogenic R160Q mutant of human sulfite oxidase (hSO) confirms the presence of three distinct species whose relative abundances depend upon pH. Species 1 is exclusively present at pH < or = 6, and remains in significant amounts even at pH 8. Variable-frequency electron spin echo envelope modulation (ESEEM) studies of this species prepared with (33)S-labeled sulfite clearly show the presence of coordinated sulfate, as has previously been found for the "blocked" form of Arabidopsis thaliana at low pH (Astashkin, A. V.; Johnson-Winters, K.; Klein, E. L.; Byrne, R. S.; Hille, R.; Raitsimring, A. M.; Enemark, J. H. J. Am. Chem. Soc. 2007, 129, 14800). The ESEEM spectra of Species 1 prepared in (17)O-enriched water show both strongly and weakly magnetically coupled (17)O atoms that can be assigned to an equatorial sulfate ligand and the axial oxo ligand, respectively. The nuclear quadrupole interaction (nqi) of the axial oxo ligand is substantially stronger than those found for other oxo-Mo(V) centers studied previously. Additionally, pulsed electron-nuclear double resonance (ENDOR) measurements reveal a nearby weakly coupled exchangeable proton. The structure for Species 1 proposed from the pulsed EPR results using isotopic labeling is a six-coordinate Mo(V) center with an equatorial sulfate ligand that is hydrogen bonded to an exchangeable proton. Six-coordination is supported by the (17)O nqi parameters for the axial oxo group of the model compound, (dttd)Mo(17)O((17)Otms), where H2dttd = 2,3:8,9-dibenzo-1,4,7,10-tetrathiadecane; tms = trimethylsilyl. Reduction of R160Q to Mo(V) with Ti(III) gives primarily Species 2, another low pH form, whereas reduction with sulfite at higher pH values gives a mixture of Species 1 and 2, as well as the "primary" high pH form of wild-type SO. The occurrence of significant amounts of the "sulfate-blocked" form of R160Q (Species 1) at physiological pH suggests that this species may be a contributing factor to the lethality of this mutation.

Cu2+Binding Modes of Recombinant α-Synuclein − Insights from EPR Spectroscopy

The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) has been proposed to play a role in Parkinson's disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-COO(-), H2O} binding persisted in conjunction with another two binding modes. Metal-ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH2, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.

Multifrequency electron spin-echo envelope modulation studies of nitrogen ligation to the manganese cluster of photosystem II

The CalEPR Center at UC-Davis (http://brittepr.ucdavis.edu) is equipped with five research grade electron paramagnetic resonance (EPR) instruments operating at various excitation frequencies between 8 and 130GHz. Of particular note for this RSC meeting are two pulsed EPR spectrometers working at the intermediate microwave frequencies of 31 and 35GHz. Previous lower frequency electron spin-echo envelope modulation (ESEEM) studies indicated that histidine nitrogen is electronically coupled to the Mn cluster in the S2 state of photosystem II (PSII). However, the amplitude and resolution of the spectra were relatively poor at these low frequencies, precluding any in-depth analysis of the electronic structure properties of this closely associated nitrogen nucleus. With the intermediate frequency instruments, we are much closer to the 'exact cancellation' limit, which optimizes ESEEM spectra for hyperfine-coupled nuclei such as 14N and 15N. Herein, we report the results from ESEEM studies of both 14N- and 15N-labelled PSII at these two frequencies. Spectral simulations were constrained by both isotope datasets at both frequencies, with a focus on high-resolution spectral examination of the histidine ligation to the Mn cluster in the S2 state.

Spectroscopic Studies of Metal Binding and Metal Selectivity in Bacillus subtilis BSco, a Homologue of the Yeast Mitochondrial Protein Sco1p

Sco1 is a mitochondrial membrane protein involved in the assembly of the CuA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco1, YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa3 (CuA-containing) oxidase activity but expressing normal levels of aa3 (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(I) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 +/- 0.01 A and by a weakly bound low-Z scatterer at 1.95 +/- 0.03 A. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 A and two O/N donor ligands at 1.95 A, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(His)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence CuA center of cytochrome c oxidase.

An ESR and ESEEM study of long-lived radicals which cause mutation in irradiated mammalian cells

Long-lived radicals (LLR) that exist in γ-ray-irradiated mammalian cells and have a very long lifetime such as 20 h induce mutation and transformation. The long-lived radicals in the irradiated mammalian cells were scavenged using AsA( L-ascorbic acid) as well as by EGCG((−)-epigallocatechin-3- O-gallate) after irradiation, accompanying the decrease in mutation in the irradiated cells. Determining the location of the long-lived radicals in the cells is therefore very important in order to elucidate the mechanism of the induction of mutation by the radicals. The electron spin echo envelope modulation (ESEEM) study indicates that the long-lived radicals are produced in the interior of the biopolymer coils where water molecules scarcely exist. Since DNA and its sugar chains are quite hydrophilic, the long-lived radicals are not produced in them. However, they are produced in proteins as a major content in cells that have several hydrophobic sites in their higher order structure. The long-lived radicals that cause mutations are not generated in DNA and/or in its sugar chains. However, they are probably produced in proteins.

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study.

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.

Structural Changes Induced in p21 Ras upon GAP-334 Complexation as Probed by ESEEM Spectroscopy and Molecular-Dynamics Simulation

Background: The means by which the protein GAP accelerates GTP hydrolysis, and thereby downregulates growth signaling by p21 Ras, is of considerable interest, particularly inasmuch as p21 mutants are implicated in a number of human cancers. A GAP “arginine finger,” identified by X-ray crystallography, has been suggested as playing the principal role in the GTP hydrolysis. Mutagenesis studies, however, have shown that the arginine can only partially account for the 10 5-fold increase in the GAP-accelerated GTPase rate of p21. Results: We report electron spin-echo envelope modulation (ESEEM) studies of GAP-334 complexed with GMPPNP bound p21 in frozen solution, together with molecular-dynamics simulations. Our results indicate that, in solution, the association of GAP-334 with GTP bound p21 induces a conformational change near the metal ion active site of p21. This change significantly reduces the distances from the amide groups of p21 glycine residues 60 and 13 to the divalent metal ion. Conclusions: The movement of glycine residues 60 and 13 upon the binding of GAP-334 in solution provides a physical basis to interpret prior mutagenesis studies, which indicated that Gly-60 and Gly-13 of p21 play important roles in the GAP-dependent GTPase reaction. Gly-60 and Gly-13 may play direct catalytic roles and stabilize the attacking water molecule and β,γ-bridging oxygen, respectively, in p21. The amide proton of Gly-60 could also play an indirect role in catalysis by supplying a crucial hydrogen bonding interaction that stabilizes loop L4 and therefore the position of other important catalytic residues.

Structural, electron paramagnetic resonance and electron spin echo envelope modulation studies of oxovanadium(iv)–amidate compounds containing monoanionic axial ligands: effect on the 51V-hyperfine coupling constants

Reaction of the amidate ligand N-{2-[(2-pyridylmethylene)amino]phenyl}pyridine-2-carbo xamide (Hcapca) with [VOCl2(thf)2] and MeONa in 1∶1∶1 molar ratio in MeCN affords the compound trans-[VOCl(capca)] 1, while reaction of 1 with NEt4NCS, MeCO2NHEt3 and imidazole yields the complexes trans-[VO(NCS)(capca)] 2, trans-[VO(Me- CO2)(capca)] 3 and trans-[VO(im)(capca)]− 4 respectively; the X-ray crystal structure of 1 is reported; Cw EPR studies of compounds 1–3 reveal a novel phenomenon of the reduction of their Az components by ca. 10% compared to N4 reference compounds.

How important is parallel-mode EPR in electron spin echo envelope modulation studies of non-Kramers systems?

Integer-spin systems with S>1 sometimes exhibit a ground-state ‘non-Kramers (NK) doublet’ in their electron paramagnetic resonance (EPR) spectra. The preferred method of studying systems with this type of EPR signal has been to use a spectrometer in which the microwave field is parallel to the applied static field (parallel-mode EPR), rather than a traditional perpendicular-mode EPR spectrometer, in order to maximize the resulting NK-EPR signal. The efficacy of parallel and perpendicular mode electron spin echo envelope modulation (ESEEM) spectroscopy on the NK doublet of azido-hemerythrin (N 3Hr red) are compared. These results demonstrate that for this technique, the advantages of parallel-mode over perpendicular-mode ESEEM are minimal at best. A simplified form of the theory underlying the analysis of NK-ESEEM is developed to explain the observations.

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II.

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.

In vivo coordination structural changes of a potent insulin-mimetic agent, bis(picolinato)oxovanadium(IV), studied by electron spin-echo envelope modulation spectroscopy

Bis(picolinato)oxovanadium(IV) [VO(pic) 2] is one of the most potent insulin-mimetic vanadium complexes. To probe coordination structural changes of this complex in vivo and provide insights into the origin of its high potency, an electron spin-echo envelope modulation (ESEEM) study was performed on organs (kidney, liver and bone) of VO(pic) 2- and VOSO 4-treated rats. Kidney and liver samples from both types of rats exhibited a 14N ESEEM signal that could be attributed to equatorially coordinating amine nitrogen. The relative intensity of the amine signal was larger for the organs of the rat treated with the less potent VOSO 4, suggesting that this amine coordination inhibits the insulin-mimetic activity. The spectra of kidney and liver from the VO(pic) 2-treated rat contained a weak signal due to the picolinate imine nitrogen. This suggests that some picolinato species (including both the bispicolinato and a partially decomposed monopicolinato species) still exist in the organs as a minor species, where the proportions of the picolinato species to the total amount of the EPR-detectable V IVO species are estimated as 8–16% in the kidney and 12–24% in the liver. The picolinate ligand presumably serves to prevent VO 2+ from being converted into the inactive amine-coordinated species. Bone samples from both types of rats exhibited an ESEEM signal due to 31P nuclei. The VO 2+ in bone is therefore most likely incorporated into the hydroxyapatite Ca 10(PO 4) 6(OH) 2 matrix, which is consistent with the hypothesis that the bone-accumulated VO 2+ is gradually released and transported to other organs as is Ca 2+. No 14N signals were observed, even in the bone samples of the VO(pic) 2-treated rats, indicating that vanadium uptake by bone requires complete decomposition of the complex.

Pulse EPR spectroscopy of Cu2+-doped inorganic glasses

Two-frequency continuous-wave and pulse EPR (electron paramagnetic resonance) spectroscopical techniques are applied to determine static and dynamic EPR parameters of Cu2+ ions in oxide and fluoride glasses. The investigations are focussed on the analysis of strain effects in the glassy matrices, the identification of the magnetic nuclei in the vicinity of Cu2+ ions as well as the determination of the dependence of the phase memory timeT M on temperature and resonance field. The results obtained by X-band continuous-wave EPR, X- and S-band echo-detected EPR, and X- and S-band electron spin echo envelope modulation studies of Cu2+-doped inorganic glasses yield information on the local symmetry of the Cu2+ coordination polyhedra, the chemical nature of the atoms in the second and higher coordination spheres, the distribution of the parameters of the static spin Hamiltonian and the low-temperature motions of the dopant-containing structural units. Special techniques like 2-D Mims ENDOR (electron nuclear double resonance) and hyperfine-correlated ENDOR are applied for the first time to doped inorganic glasses. From the spin relaxation measurements a stronger tendency of the Cu2+ ions to aggregate is found for fluoride glasses in comparison to aluminosilicate and phosphate glasses.

High-Frequency/High-Field Electron Spin Echo Envelope Modulation Study of Nitrogen Hyperfine and Quadrupole Interactions on a Disordered Powder Sample

High-frequency/high-field (95 GHz/3.4 T) electron spin echo envelope modulation (ESEEM) experiments on single crystals and disordered samples of dianisyl-nitroxide (DANO) radicals are reported. At these high microwave frequencies (W-band), the anisotropicg-matrix of the nitroxide radical is resolved in the EPR spectrum. Additionally ESEEM modulations from other than nitrogen nuclei, such as protons, are highly suppressed at these frequencies, because they are too far from the cancellation condition for effective mixing of the nuclear spin functions. Therefore the nitrogen (14N) hyperfine and quadrupole coupling tensors could be determined without ambiguity from powder measurements. The results obtained were checked by ESEEM measurements on single crystals. Advantages and disadvantages of high-field ESEEM on nitrogen couplings are briefly discussed and compared with electron nuclear double resonance (ENDOR) andX-band ESEEM.

A comparative electron spin echo envelope modulation study of the primary electron acceptor quinone in Zn-substituted and cyanide-treated preparations of photosystem II

The electron spin echo envelope modulation spectra of the reduced primary acceptor quinone, QA, in two preparations of plant photosystem II, have been studied. In one of these preparations the Fe2+ ion in the quinone–iron complex has been substituted by diamagnetic Zn2+. In the other preparation this iron ion has been converted into the diamagnetic state using a potassium cyanide treatment. A comparative analysis of two-dimensional three-pulse electron spin echo envelope modulation spectra has shown similar structure of the binding site of QA in both preparations. Two nitrogen nuclei have been found to contribute to the spectra in both preparations. One of these nitrogens is, most probably, an amino nitrogen in the imidazole ring of histidine 215 of the D2 protein. The other nitrogen has been assigned to the peptide group of alanine 261 of the D2 protein. The numerical simulations of the electron spin echo envelope modulation spectra have shown that both nitrogens are simultaneously bound to QA.