Electron Spin Echo Envelope Modulation Patterns Research Articles

Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Previously, an electron spin echo envelope modulation (ESEEM) spectroscopic approach was established to probe the local secondary structure of membrane proteins and peptides utilizing site-directed spin-labeling (SDSL). In this method, the side chain of one amino acid residue is selectively 2H-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the 2H-labeled amino acid (denoted as i ± 1 to i ± 4, i represents the 2H-labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and 2H atoms on the amino acid side chain. Due to the periodicity of different secondary structures, different ESEEM patterns can be revealed to probe the structure. For an α-helical structural component, a 2H ESEEM signal can be detected for i ± 3 and i ± 4 samples, but not for i ± 1 or i ± 2 samples. Several 2H-labeled hydrophobic amino acids have been demonstrated in model system that can be utilized to identify local secondary structures via this ESEEM approach in an extremely efficient fashion. In this study, the ESEEM approach was used to investigate the rod 2B region of the full-length intermediate filament protein human vimentin. Consistent with previous EPR and X-ray crystallography results, our ESEEM results indicated helical structural components within this region. Thus, this ESEEM approach is able to identify α-helical structural components despite the coiled-coil nature of the vimentin structure. The data show that the human vimentin rod 2B adapted a typical α-helical structure around residue Leu309. This result is consistent with the X-ray data from fragmented protein segments and continuous wave EPR data on the full-length vimentin. Finally, the ESEEM data suggested that a local secondary structure slightly different from a typical α-helix was adopted around residue 340.

Orientation Information from the Dipolar Interaction Between a Complex in the Excited Quartet State and a Doublet Spin Label

An algorithm is proposed for deriving the position of a stable radical relative to a photoexcited quartet state from the electron spin–spin interactions measured by double resonance methods. Intersystem crossing generates multiplet polarization in the quartet state and microwave excitation of the ±3/2 ↔ ±1/2 transitions converts the multiplet polarization into net polarization of the ±1/2 levels. The dependence of the electron spin echo envelope modulation (ESEEM) of the +1/2 ↔ −1/2 transition on the field/frequency of the stimulation pulse is demonstrated. The algorithm is tested by comparing the predicted ESEEM patterns to those from explicit numerical calculations of the spin evolution (so-called numerical experiments), which act as a model for experiment results. The comparison demonstrates that within the point-dipole approximation it is feasible to obtain not only the distance between the two paramagnetic centers but also the orientation of the distance vector relative to the principal axes of the quartet state.

Elucidation of coordination structure aroundCe3+in dopedSiO2glasses using pulsed electron paramagnetic resonance: Effect of phosphorus, boron, and phosphorus-boron codoping

Effects of codoping of phosphorus and/or boron on the coordination sphere of a ${\mathrm{Ce}}^{3+}$ in ${\mathrm{SiO}}_{2}$ glasses were examined by applying pulsed electron paramagnetic resonance (EPR) electron spin echo envelope modulation (ESEEM) spectroscopy. Simulation of the observed ESEEM pattern revealed a striking difference in the solvation shell structure between the P-doped and the P-free glasses, i.e., the P doping is very effective for the formation of the solvation shell, while no such an effect was observed for the B doping. When P and B are simultaneously doped, the solvation shell structure is close to that in the P-doped samples. Photoluminescence properties of ${\mathrm{Ce}}^{3+}$ in these samples were found to be classified into two categories corresponding to the solvation shell structures.

Superoxide Ion Encaged in Nanoporous Crystal 12CaO·7Al2O3 Studied by Continuous Wave and Pulsed Electron Paramagnetic Resonance

Continuous wave and pulsed electron paramagnetic resonance studies were performed on the superoxide ion, O 2 - , encaged in the nanoporous crystal 12CaO.7Al 2 O 3 (C12A7). Hyperfine splitting due to 1 7 O in O 2 - was observed in C12A7 heated in 40% 1 7 O-enriched O 2 gas at 550 °C, providing solid evidence that encaged O 2 derives from gaseous O 2 in the atmosphere via reactions with free oxygen ions in the cage. Angular variations of g values, 1 7 O hyperfine splitting in the single crystal at 20 K, and 2 7 Al electron spin-echo envelope modulation pattern for the powder at 4 K clarified that an O 2 - is encaged in a subnanometer-sized cage of C12A7 and adsorbed on a Ca 2 + ion forming a part of the cage wall. Further, two oxygen atoms in the O 2 - ion are equivalent in the cage, indicating that the O 2 - ion takes a side-on configuration. Then, the O-O bond is perpendicular to the 2-fold rotation axis (C 2 ∥ (100)) across the center of the cage and directed to two oxygen ions forming the cage wall. Variation in the g values with temperature indicates that the O 2 - may be regarded as solidlike below ∼20 K and above this temperature the rocking motion of the O-O bond about the C 2 axis is activated with an increase in temperature, but the anisotropy in the motion remains even at 400 K. This result may be explained by a view that Coulombic interaction of the Ca 2 + with the fully occupied π orbital in the O 2 - is stronger than that with the semioccupied π orbital, controlling the dynamics.

High-affinity metal-binding site in beef heart mitochondrial F1ATPase: an EPR spectroscopy study.

The high-affinity metal-binding site of isolated F(1)-ATPase from beef heart mitochondria was studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using Mn(II) as a paramagnetic probe. The protein F(1) was fully depleted of endogenous Mg(II) and nucleotides [stripped F(1) or MF1(0,0)] and loaded with stoichiometric Mn(II) and stoichiometric or excess amounts of ADP or adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP). Mn(II) and nucleotides were added to MF1(0,0) either subsequently or together as preformed complexes. Metal-ADP inhibition kinetics analysis was performed showing that in all samples Mn(II) enters one catalytic site on a beta subunit. From the HF-EPR spectra, the zero-field splitting (ZFS) parameters of the various samples were obtained, showing that different metal-protein coordination symmetry is induced depending on the metal nucleotide addition order and the protein/metal/nucleotide molar ratios. The electron spin-echo envelope modulation (ESEEM) technique was used to obtain information on the interaction between Mn(II) and the (31)P nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured (31)P hyperfine couplings clearly indicated coordination changes related to the metal nucleotide addition order and the protein/metal/nucleotide ratios. On the contrary, the samples with AMPPNP showed very similar ESEEM patterns, despite the remarkable differences present among their HF-EPR spectra. This fact has been attributed to changes in the metal-site coordination symmetry because of ligands not involving phosphate groups. The kinetic data showed that the divalent metal always induces in the catalytic site the high-affinity conformation, while EPR experiments in frozen solutions supported the occurrence of different precatalytic states when the metal and ADP are added to the protein sequentially or together as a preformed complex. The different states evolve to the same conformation, the metal(II)-ADP inhibited form, upon induction of the trisite catalytic activity. All our spectroscopic and kinetic data point to the active role of the divalent cation in creating a competent catalytic site upon binding to MF1, in accordance with previous evidence obtained for Escherichia coli and chloroplast F(1).

Direct Observation of Electron Spin Density on TDAE Cations in the Ferromagnetic State of SolidTDAE−C60

${}^{14}\mathrm{N}$ electron spin echo envelope modulation (ESEEM) and ${}^{1}\mathrm{H}$ nuclear magnetic resonance (NMR) techniques have been employed in order to measure the unpaired electron spin density of amino nitrogens and ${\mathrm{CH}}_{3}$ protons, in the low temperature ferromagnetic state of solid $\mathrm{TDAE}\ensuremath{-}{\mathrm{C}}_{60}$. Well resolved ${}^{14}\mathrm{N}$ ESEEM patterns were obtained for the first time below 9 K, and their analysis reveals an extremely low unpaired electron spin density on specific TDAE nitrogen sites. Also, ${}^{1}\mathrm{H}$ NMR line shape measurements corroborate a stepwise increase of the electron spin density on the ${\mathrm{CH}}_{3}$ protons below 9 K that is accompanied by the onset of an activated law for ${}^{1}\mathrm{H}$ $1/{T}_{1}$ with temperature.

14N electron spin-echo envelope modulation of the S = 3/2 spin system of the Azotobacter vinelandii nitrogenase iron-molybdenum cofactor.

Wild-type nitrogenase MoFe protein shows a deep 14N electron spin-echo envelope modulation (ESEEM) arising from a nitrogen nucleus (N1) coupled to the S = 3/2 spin system of the FeMo-cofactor of the MoFe protein. A previous ESEEM study on altered MoFe proteins generated by substitutions at the alpha-195-histidine position suggested that alpha-195-histidine provides a hydrogen bond to the FeMo-cofactor but is not the source of the 14N1 modulation [DeRose et al. (1995) Biochemistry 34, 2809-2814]. This study also raised the possibility of a correlation between ESEEM spectroscopic properties and the nitrogenase phenotype. We now report ESEEM studies on altered MoFe proteins with substitutions at residues alpha-96-arginine, alpha-359-arginine, and alpha-381-phenylalanine to (i) assign the first-shell hydrogen bonding as revealed by the 14N modulation; (ii) explore the mechanistic relevance of the ESEEM signatures to nitrogenase activity; and (iii) study microscopic changes within the polypeptide environment of the FeMo-cofactor. Present ESEEM data reveals that two kinds of 14N modulations are present in wild-type MoFe protein. A new 2-dimensional procedure for high-precision analysis of the ESEEM data of the MoFe proteins shows that the deep wild-type ESEEM modulation (denoted N1) has a hyperfine-coupling constant of Aiso = 1.05 MHz and nuclear quadrupole coupling parameters of e2qQ = 2.17 MHz, eta = 0.59; the other (denoted N2) has a smaller hyperfine coupling of Aiso = approximately 0.5 MHz and e2qQ = approximately 3.5 MHz, eta = approximately 0.4. The N2 ESEEM pattern is more obvious when unmasked by substitutions that result in the loss of the deep N1 modulation. Correlations of the ESEEM properties and catalytic activities of the altered MoFe proteins suggest that (i) the side chain of the alpha-359-arginine is the source of the deep ESEEM N1 modulation; (ii) one or both of the amide nitrogens of alpha-356-glycine/alpha-357-glycine are responsible for the weak N2 modulation; (iii) substitution of the nonpolar alpha-381-phenylalanine residue, as well as substitution of either the alpha-195-histidine or alpha-359-arginine residues, can eliminate the N1 interaction with FeMo-cofactor; and (iv) ESEEM can be used to detect slight reorientations of FeMo-cofactor within its polypeptide pocket, although the mechanistic relevance of the loss or perturbation of the hydrogen-bonding interactions between FeMo-cofactor and polypeptide environment has not yet been established.

Interaction between Starburst Dendrimers and SDS micelles studied by continuous-wave and pulsed electron spin resonances

A computer aided analysis of both cw-and pulsed-Electron Paramagnetic Resonance (EPR) spectra of 5doxylstearic acid (5DXSA) as a probe was carried out to compare the aggregation process of Sodium Dodecyl Sulfate (SDS) surfactants in the absence and in the presence of Starburst Dendrimers (SBDs), and to provide information on the interactions between SDS and SBDs. Mobility and polarity parameters were extracted from the cw-EPR analysis whereas the analysis of the Electron Spin Echo Envelope Modulation (ESEEM) signal provided details about the doxyl environment in the SDS micelles. In the absence of SBDs, the formation of SDS micelles was revealed by the decrease in mobility of the probes inserted in the micelles. The high packing of SDS chains in the micelles prevented the water permeability at the doxyl site. In the presence of the dendrimers, the analysis of the EPR spectra suggested the formation of SDS aggregates at the dendrimer surface (cooperative interaction). The larger the size of the dendrimers and the protonation of their surface, the stronger the interactions resulted between the SDS surfactants and the SBD surface. The analysis of the ESEEM pattern indicated that the cooperative interaction of the surfactant with the SBD surface led to a less packed structure of the aggregates. A schematic view was proposed to describe the local structure of the doxyl group and its environment in the absence and in the presence of the dendrimers.

Coordination of the Rieske-type [2Fe-2S] cluster of the terminal iron-sulfur protein of Pseudomonas putida benzene 1,2-dioxygenase, studied by one- and two-dimensional electron spin-echo envelope modulation spectroscopy.

One- and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the [2Fe-2S] cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex. The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from gz through to gx) are consistent with their assignment to two 14N nuclei. Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two 14N nuclei. On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these 14N nuclei [N(1), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz], the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster. Bonding parameters of the two [14N]histidine ligands were calculated from these hyperfine couplings. The primary covalent contributions to the hyperfine interaction arise from 14N-to-Fe2+ sigma bonds. For N(1), our analysis of the percentage of unpaired 2s and 2p electrons gave f2s approximately 1.3% and f2p approximately 0.2%, while values of f2s approximately 1.7% and f2p approximately 1.4% were estimated for N(2). Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type [2Fe-2S] center of Pseudomonas cepacia phthalate dioxygenase [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871] indicates an apparent reduction in unpaired electron spin density residing on the two 14N ligands of ISPBED. Analysis of slices of the HYSCORE spectrum has provided evidence for another 14N nucleus (A approximately 1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type [2Fe-2S] clusters. This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers. The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers.

Two-Dimensional ESEEM Study of VO2+ Complexes with Imidazole and Histidine: Histidine Is a Polydentante Ligand

The oxovanadium cation, VO{sup 2+}, has been successfully employed as a spin probe in electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) studies of metalloprotein active sites. Here we demonstrate that the complex of VO{sup 2+} with histidine has been misinterpreted and that the dominant ESEEM pattern is produced by the {alpha}-amino group. Nevertheless, we show that histidine side chain ligands can be identified from either nitrogen hyperfine or quadrupole couplings. The application of 2D ESEEM to the [{sup 15}N]imidazole complex shows, in the same spectrum, both the coordinated and the remote ring nitrogens with very different isotropic hyperfine couplings. In the case of histidine ligation, equatorial coordination by both imine and amine nitrogen is demonstrated. The reproducible differences between both the hyperfine and the quadrupole constants of the amine and imine nitrogens are reflected in the shape and position of the lines in the 2D spectra and allow them to be distinguished in proteins. With both imine and amine coordination, the VO{sup 2+}/histidine complex cannot be considered an adequate model for coordination by the histidine side chain in proteins. The imidazole, as a monodentante ligand, is better for this role. 16 refs., 3 figs.

Electron spin-echo envelope modulation studies of the [3Fe–4S] cluster of Escherichia coli fumarate reductase

The dimeric form of purified Escherichia coli fumarate reductase contains three different iron–sulfur clusters, termed centres 1, 2 and 3 ([2Fe–2S], [4Fe–4S] and [3Fe–4S] clusters, respectively). We have performed electron spin-echo envelope modulation (ESEEM) spectroscopy in order to obtain information about the environment of the [3Fe–4S] cluster. Modulations from 14N were detected in the three-pulse ESEEM spectra from the cluster in the oxidised state. The low-frequency lines observed in the three-pulse ESEEM pattern were similar to those observed for the [3Fe–4S] cluster of bovine heart succinate dehydrogenase (B. A. C. Ackrell, E. B. Kearney, W. B. Mims, J. Peisach and J. H. Beinert, J. Biol. Chem., 1984, 259, 4015). Determination of the hyperfine and quadrupolar coupling (A≈ 0.7 MHz, e2qQ≈ 3.4 MHz) gave values that are similar to those determined for the [2Fe–2S] cluster of centre 1 (R. Cammack, A. Chapman, J. McCracken, J. B. Cornelius, J. Peisach and J. H. Weiner, Biochim. Biophys. Acta, 1988, 956, 307; J. K. Shergill, PhD thesis, 1993). These coupling constants indicate that the 14N-nucleus giving rise to the modulation pattern in the ESEEM spectrum is weakly coupled to centre 3, rather than directly coordinated to the iron.

Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske iron-sulfur centers of the cytochrome b6f complex on spinach and the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria

Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b6f complex of spinach chloroplasts and of the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of 14N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b6f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A1 = 4.6 MHz and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc1 complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic coupling to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferredoxin ESEEM patterns.

Optically detected low field electron spin echo envelope modulations of fluorescent N-V centers in diamond

For the diamond N-V center in its 3A ground state distinct oscillatory behavior is observed in optically detected two-pulse and stimulated echo decays upon the application of low magnetic fields ( H⪢200 G). These electron spin echo envelope modulation (ESEEM) effects are found to result from dipole-dipole interactions between the triplet spin of the axially symmetric N-V center, and 13C ( I = 1 2 ) nuclear spins, distributed randomly in the diamond lattice. The ESEEM patterns are discussed to be very sensitive to the applied magnetic fields. A kilohertz resolution of the nuclear induced splittings of the N-V center is reached in the experiments.

A theoretical treatment of multiple quantum nuclear spin coherences in electron spin echo studies of polyacetylene

We present a theoretical investigation of the origin of the unusual 3-pulse electron spin echo envelope modulation (ESEEM) pattern obtained from 13C enriched polyacetylene, which exhibits modulation at multiple harmonics of the nuclear Larmor frequency ωn. ESEEM signals have been calculated for different models of the electron–nuclear spin coupling. It is shown that the local hyperfine tensors observed in electron nuclear double resonance (ENDOR) experiments on (13CH)x cannot account for the ESEEM results. These tensors have isotropic components which are too large to give narrow peaks at ωn, and there are too few nuclei to produce the large number of harmonics observed. Simulations for a point electron coupled to nuclei on a spherical shell, the spherical model, give insight into the conditions required to obtain narrow lines at multiple harmonics of ωn: The electron must be weakly coupled to a very large number of nuclei. Analytical results show that for the spherical model in the limit of small coupling, the intensity profile of the Fourier transform of the three-pulse ESEEM will be a Gaussian of width k̄ N, where k̄ is a function of the magnitude of the electron–nuclear coupling and N is the number of nuclei. Simulations assuming that the unpaired electron is delocalized in one dimension along the polyene chain and dipolar coupled to the 13C nuclei on neighboring chains give multiple harmonic patterns, at a reduced interchain distance. We conclude that the observed ESEEM pattern arises from nuclei on neighboring polyacetylene chains and is closely related to the matrix/distant ENDOR line observed for (13CH)x.

Enhancement of nuclear modulation in electron spin echoes at low magnetic fields: S-band ESE spectrometer

Most electron spin echo (ESE) experiments are performed at X-band (≈ 9 GHz) microwave frequencies. Just as in cw EPR, however, a multifrequency approach to ESE spectroscopy provides a more accurate determination of spin Hamiltonian parameters. The use of lower microwave frequencies (fields) in ESE experiments is expected to be particularly useful for enhancement of electron spin echo envelope modulation (ESEEM) depth for weakly coupled nuclei. Therefore, a pulsed EPR spectrometer was constructed to operate at S-band (2–4 GHz). Comparisons of vitrinite, perylene cation, irradiated quartz, and complexed Gd(III) ESEEM patterns at X- and S-band illustrate the expected modulation depth enhancement.