Publications

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long-time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron-electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.

The electron–electron double resonance (DEER) method, which provides distance distributions between two spin labels, attached site specifically to biomolecules (proteins and nucleic acids), is currently a well-recognized biophysical tool in structural biology. The most commonly used spin labels are based on nitroxide stable radicals, conjugated to the proteins primarily via native or engineered cysteine residues. However, in recent years, new spin labels, along with different labeling chemistries, have been introduced, driven in part by the desire to study structural and dynamical properties of biomolecules in their native environment, the cell. This mini-review focuses on these new spin labels, which allow for DEER on orthogonal spin labels, and on the state of the art methods for in-cell DEER distance measurements.

This is a methodological guide to the use of deep neural networks in the processing of pulsed dipolar spectroscopy (PDS) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs and paramagnetic metal ions. PDS uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their “robust black box” reputation hides the complexity of their design and training – particularly when the training dataset is effectively infinite. The objective of this paper is to give insight into training against simulated databases, to discuss network architecture choices, to describe options for handling DEER (double electron-electron resonance) and RIDME (relaxation-induced dipolar modulation enhancement) experiments, and to provide a practical data processing flowchart.

Hsp90 is an important molecular chaperone that facilitates the maturation of client proteins. It is a homodimer, and its function depends on a conformational cycle controlled by ATP hydrolysis and co-chaperones binding. We explored the binding of co-chaperone Sba1 to yeast Hsp90 (yHsp90) and the associated conformational change of yHsp90 in the pre- and post-ATP hydrolysis states by double electron-electron resonance (DEER) distance measurements. We substituted the Mg(II) cofactor at the ATPase site with paramagnetic Mn(II) and established the binding of Sba1 by measuring the distance between Mn(II) and a nitroxide (NO) spin-label on Sba1. Then, Mn(II)-NO DEER measurements on yHsp90 labeled with NO at the N-terminal domain detected the shift toward the closed conformation for both hydrolysis states. Finally, Mn(II)-Mn(II) DEER showed that Sba1 induced a closed conformation different from those with just bound Mn(II)·nucleotides. Our results provide structural experimental evidence for the binding of Sba1 tuning the closed conformation of yHsp90.

This is a methodological guide to the use of deep neural networks in the processing of double electron-electron resonance (DEER, aka PELDOR) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs. DEER spectroscopy uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their "robust black box" reputation hides the complexity of their design and training - particularly when the training dataset is effectively infinite. The objective of this paper is to give insight into training against infinite databases, to shed some light on the processes inside the neural net, and to provide a practical data processing flowchart for structural biology work.

Double electron–electron resonance (DEER) is a pulse electron paramagnetic resonance (EPR) technique that measures distances between paramagnetic centres. It utilizes a four-pulse sequence based on the refocused Hahn spin echo. The echo decays with increasing pulse sequence length 2(τ1+τ2) , where τ1 and τ2 are the two time delays. In DEER, the value of τ2 is determined by the longest inter-spin distance that needs to be resolved, and τ1 is adjusted to maximize the echo amplitude and, thus, sensitivity. We show experimentally that, for typical spin centres (nitroxyl, trityl, and Gd(III)) diluted in frozen protonated solvents, the largest refocused echo amplitude for a given τ2 is obtained neither at very short τ1 (which minimizes the pulse sequence length) nor at τ1=τ2 (which maximizes dynamic decoupling for a given total sequence length) but rather at τ1 values smaller than τ2 . Large-scale spin dynamics simulations based on the coupled cluster expansion (CCE), including the electron spin and several hundred neighbouring protons, reproduce the experimentally observed behaviour almost quantitatively. They show that electron spin dephasing is driven by solvent protons via the flip-flop coupling among themselves and their hyperfine couplings to the electron spin.

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant K-D was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. This method provides the distance distribution between two spin labels attached at specific, well-defined positions in a protein. For the method to be viable under in-cell conditions, the spin label and its attachment to the protein should exhibit high chemical stability in the cell. Here we present low-temperature, trityl-trityl DEER distance measurements on two model proteins, PpiB (prolyl cis-trans isomerase from E. coli) and GB1 (immunoglobulin G-binding protein), doubly labeled with the trityl spin label, CT02MA. Both proteins gave in-cell distance distributions similar to those observed in vitro, with maxima at 4.5-5 nm, and the data were further compared with in-cell Gd(III)-Gd(III) DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. These results highlight the challenges of designing trityl tags suitable for in-cell distance determination at ambient temperatures on live cells.

Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(III). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(III) tag was achieved as well and the trityl-Gd(III) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(III)-Gd(III) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(III)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(III)/ trityl labelling schemes.

Dynamic Nuclear Polarization (DNP) is an efficient technique for enhancing NMR signals by utilizing the large polarization of electron spins to polarize nuclei. The mechanistic details of the polarization transfer process involve the depolarization of the electrons resulting from microwave (MW) irradiation (saturation), as well as electron-electron cross-relaxation occurring during the DNP experiment. Recently, electron-electron double resonance (ELDOR) experiments have been performed under DNP conditions to map the depolarization profile along the EPR spectrum as a consequence of spectral diffusion. A phenomenological model referred to as the eSD model was developed earlier to describe the spectral diffusion process and thus reproduce the experimental results of electron depolarization. This model has recently been supported by quantum mechanical calculations on a small dipolar coupled electron spin system, experiencing dipolar interaction based cross-relaxation. In the present study, we performed a series of ELDOR measurements on a solid glassy solution of TEMPOL radicals in an effort to substantiate the eSD model and test its predictability in terms of electron depolarization profiles, in the steady-state and under non-equilibrium conditions. The crucial empirical parameter in this model is (eSD), which reflects the polarization exchange rate among the electron spins. Here, we explore further the physical basis of this parameter by analyzing the ELDOR spectra measured in the temperature range of 3-20 K and radical concentrations of 20-40 mM. Simulations using the eSD model were carried out to determine the dependence of (eSD) on temperature and concentration. We found that for the samples studied, (eSD) is temperature independent. It, however, increases with a power of approximate to 2.6 of the concentration of TEMPOL, which is proportional to the average electron-electron dipolar interaction strength in the sample.

Distance measurements by electron-electron double resonance (DEER) carried out on spin-labeled proteins delivered into cells provide new insights into the conformational states of proteins in their native environment. Such measurements depend on spin labels that exhibit high redox stability and high DEER sensitivity. Here we present a new Gd(III)-based spin label, BrPSPy-DO3A-Gd(III), which was derived from an earlier label, BrPSPy-DO3MA-Gd(III), by removing the methyl group from the methyl acetate pending arms. The small chemical modification led to a reduction in the zero-field splitting and to a significant increase in the phase memory time, which together culminated in a remarkable improvement of in-cell DEER sensitivity, while maintaining the high distance resolution. The excellent performance of BrPSPy-DO3A-Gd(III) in in-cell DEER measurements was demonstrated on doubly labeled ubiquitin and GB I delivered into HeLa cells by electroporation.

Mn2+ often serves as a paramagnetic substitute to Mg2+, providing means for exploring the close environment of Mg2+ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg2+-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band 31P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) – detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring 15N-31P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn2+ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for 55Mn-31P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with 15N-31P correlations.

High-affinity chelating tags for Gd(III) and Mn(II) ions that provide valuable high-resolution distance restraints for biomolecules were used as spin labels for double electron-electron resonance (DEER) measurements. The availability of a generic tag that can bind both metal ions and provide a narrow and predictable distance distribution for both ions is attractive owing to their different EPR-related characteristics. Herein we introduced two paramagnetic tags, 4PSPyMTA and 4PSPyNPDA, which are conjugated to cysteine residues through a stable thioether bond, forming a short and, depending on the metal ion coordination mode, a rigid tether with the protein. These tags exhibit high affinity for both Mn(II) and Gd(III) ions. The DEER performance of the 4PSPyMTA and 4PSPyNPDA tags, in complex with Gd(III) or Mn(II), was evaluated for three double cysteine mutants of ubiquitin, and the Gd(III)-Gd(III) and Mn(II)-Mn(II) distance distributions they generated were compared. All three Gd(III) complexes of the ubiquitin-PyMTA and ubiquitin-PyNPDA conjugates produced similar and expected distance distributions. In contrast, significant variations in the maxima and widths of the distance distributions were observed for the Mn(II) analogs. Furthermore, whereas PyNPDA-Gd(III) and PyNPDA-Mn(II) delivered similar distance distributions, appreciable differences were observed for two mutants with PyMTA, with the Mn(II) analog exhibiting a broader distance distribution and shorter distances. ELDOR (electron-electron double resonance)-detected NMR measurements revealed some distribution in the Mn(II) coordination environment for the protein conjugates of both tags but not for the free tags. The broader distance distributions generated by 4PSPyMTA-Mn(II), as compared with Gd(III), were attributed to the distributed location of the Mn(II) ion within the PyMTA chelate owing to its smaller size and lower coordination number that leave the pyridine nitrogen uncoordinated. Accordingly, in

Double-arm cyclen-based Gd3+ tags are shown to produce accurate nanometer scale Gd3+-Gd3+ distance measurements in double electron-electron resonance (DEER) experiments by confining the space accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an alpha-helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd3+-Gd3+ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double-arm Gd3+ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity.

Gd3+-based spin labels are useful as an alternative to nitroxides for intramolecular distance measurements at high fields in biological systems. However, double electron-electron resonance (DEER) measurements using model Gd3+ complexes featured a low modulation depth and an unexpected broadening of the distance distribution for short Gd3+-Gd3+ distances, when analysed using the software designed for S = 1/2 pairs. It appears that these effects result from the different spectroscopic characteristics of Gd3+-the high spin, the zero field splitting (ZFS), and the flip-flop terms in the dipolar Hamiltonian that are often ignored for spin-1/2 systems. An understanding of the factors affecting the modulation frequency and amplitude is essential for the correct analysis of Gd3+-Gd3+ DEER data and for the educated choice of experimental settings, such as Gd3+ spin label type and the pulse parameters. This work uses time-domain simulations of Gd3+-Gd3+ DEER by explicit density matrix propagation to elucidate the factors shaping Gd3+ DEER traces. The simulations show that mixing between the vertical bar+1/2, -1/2 > and vertical bar-1/2, +1/2 > states of the two spins, caused by the flip-flop term in the dipolar Hamiltonian, leads to dampening of the dipolar modulation. This effect may be mitigated by a large ZFS or by pulse frequency settings allowing for a decreased contribution of the central transition and the one adjacent to it. The simulations reproduce both the experimental line shapes of the Fourier-transforms of the DEER time domain traces and the trends in the behaviour of the modulation depth, thus enabling a more systematic design and analysis of Gd3+ DEER experiments. Published by AIP Publishing.

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (mRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 30,50-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the mRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L reversible arrow AO + L reversible arrow BO reversible arrow BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved mRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling is a very powerful tool for elucidating the structure and organization of biomolecules. Gd3+ complexes have recently emerged as a new class of spin labels for distance determination by pulsed EPR spectroscopy at Q-and W-band. We present CW EPR measurements at 240 GHz (8.6 Tesla) on a series of Gd-rulers of the type Gd-PyMTA-spacer-Gd-PyMTA, with Gd-Gd distances ranging from 1.2 nm to 4.3 nm. CW EPR measurements of these Gd-rulers show that significant dipolar broadening of the central |-1/2 >->|1/2 > transition occurs at 30 K for Gd-Gd distances up to similar to 3.4 nm with Gd-PyMTA as the spin label. This represents a significant extension for distances accessible by CW EPR, as nitroxide-based spin labels at X-band frequencies can typically only access distances up to similar to 2 nm. We show that this broadening persists at biologically relevant temperatures above 200 K, and that this method is further extendable up to room temperature by immobilizing the sample in glassy trehalose. We show that the peak-to-peak broadening of the central transition follows the expected 1/r(3) dependence for the electron-electron dipolar interaction, from cryogenic temperatures up to room temperature. A simple procedure for simulating the dependence of the lineshape on interspin distance is presented, in which the broadening of the central transition is modeled as an S = 1/2 spin whose CW EPR lineshape is broadened through electron-electron dipolar interactions with a neighboring S = 7/2 spin.

By providing accurate distance measurements between spin labels site-specifically attached to bio-macromolecules, double electron-electron resonance (DEER) spectroscopy provides a unique tool to probe the structural and conformational changes in these molecules. Gd3+-tags present an important family of spin-labels for such purposes, as they feature high chemical stability and high sensitivity in high-field DEER measurements. The high sensitivity of the Gd3+ ion is associated with its high spin (S = 7/2) and small zero field splitting (ZFS), resulting in a narrow spectral width of its central transition at high fields. However, under the conditions of short distances and exceptionally small ZFS, the weak coupling approximation, which is essential for straightforward DEER data analysis, becomes invalid and the pseudo-secular terms of the dipolar Hamiltonian can no longer be ignored. This work further explores the effects of pseudo-secular terms on Gd3+-Gd3+ DEER measurements using a specifically designed ruler molecule; a rigid bis-Gd3+-DOTA model compound with an expected Gd3+-Gd3+ distance of 2.35 nm and a very narrow central transition at the W-band (95 GHz). We show that the DEER dipolar modulations are damped under the standard W-band DEER measurement conditions with a frequency separation, Delta nu, of 100 MHz between the pump and observe pulses. Consequently, the DEER spectrum deviates considerably from the expected Pake pattern. We show that the Pake pattern and the associated dipolar modulations can be restored with the aid of a dual mode cavity by increasing Delta nu from 100 MHz to 1.09 GHz, allowing for a straightforward measurement of a Gd3+-Gd3+ distance of 2.35 nm. The increase in Dn increases the contribution of the vertical bar-5/2 > -> vertical bar-3/2 > and vertical bar-7/2 > -> vertical bar-5/2 > transitions to the signal at the expense of the vertical bar-3/2 > -> vertical bar-1/2 > transition, thus minimizing the effect of dipolar pseudo-secul

Complexes of the Gd(III) ion are currently being established as spin labels for distance determination in biomolecules by pulse dipolar spectroscopy. Because Gd(III) is an f ion, one expects electron spin density to be localized on the Gd(III) ion - an important feature for the mentioned application. Most of the complex ligands have nitrogens as Gd(III) coordinating atoms. Therefore, measurement of the (14)N hyperfine coupling gives access to information on the localization of the electron spin on the Gd(III) ion. We carried out W-band, 1D and 2D (14)N and (1)H ENDOR measurements on the Gd(III) complexes Gd-DOTA, Gd-538, Gd-595, and Gd-PyMTA that serve as spin labels for Gd-Gd distance measurements. The obtained (14)N spectra are particularly well resolved, revealing both the hyperfine and nuclear quadrupole splittings, which were assigned using 2D Mims ENDOR experiments. Additionally, the spectral contributions of the two different types of nitrogen atoms of Gd-PyMTA, the aliphatic N atom and the pyridine N atom, were distinguishable. The (14)N hyperfine interaction was found to have a very small isotropic hyperfine component of -0.25 to -0.37MHz. Furthermore, the anisotropic hyperfine interactions with the (14)N nuclei and with the non-exchangeable protons of the ligands are well described by the point-dipole approximation using distances derived from the crystal structures. We therefore conclude that the spin density is fully localized on the Gd(III) ion and that the spin density distribution over the nuclei of the ligands is rightfully ignored when analyzing distance measurements.

Gd3+ tags have been shown to be useful for performing distance measurements in biomolecules via the double electron electron resonance (DEER) technique at Q- and W-band frequencies. We introduce a new cyclen-based Gd3+ tag that exhibits a relatively narrow electron paramagnetic resonance (EPR) spectrum, affording high sensitivity, and which yields exceptionally narrow Gd3+-Gd3+ distance distributions in doubly tagged proteins owing to a very short tether. Both the maxima and widths of distance distributions measured for tagged mutants of the proteins ERp29 and T4 lysozyme, featuring Gd3+-Gd3+ distances of ca. 6 and 4 nm, respectively, were well reproduced by simulated distance distributions based on available crystal structures and sterically allowed rotamers of the tag. The precision of the position of the Gd3+ ion is comparable to that of the nitroxide radical in an MTSL-tagged protein and thus the new tag represents an attractive tool for performing accurate distance measurements and potentially probing protein conformational equilibria.

Applications of distance measurements by pulse dipolar electron-paramagnetic resonance (PD-EPR) spectroscopy to structural biology are based on introducing spin labels (SLs) at well-defined locations in the biomacromolecule. The most commonly used SLs are nitroxyl radicals, but recently SLs based on high-spin Gd(3+) (S=7/2) complexes have been shown to be an attractive alternative for PD-EPR, particularly double electron-electron resonance (DEER), at spectrometer frequencies higher than 30GHz. In this chapter, we describe the advantage of using this new family of SLs in terms of sensitivity, stability, and chemical diversity. We present current labeling strategies for proteins, discuss the approximations under which DEER data analysis of a pair of Gd(3+) SLs (GdSLs) is equivalent to that of a pair of S=1/2 SLs, and discuss the reduction in multispin effects in a cluster of GdSLs, as opposed to a cluster of nitroxide labels, which can be found in oligomeric systems. In addition, we provide a brief overview of the current, rather limited, knowledge of Gd(3+) phase relaxation behavior and describe experimental strategies in terms of optimizing sensitivity. The possibility of using several types of SLs in a system allows one to isolate effects due to the chemical nature of the SL itself; several such examples are presented, focusing on comparing nitroxide and GdSLs. Finally, we will discuss the initial results on in-cell DEER with GdSLs.

Mn2+ localization in hairpin 92 of the 23S ribosomal RNA (HP92) was obtained using W-band (95 GHz) DEER (double electron-electron resonance) distance measurements between the Mn2+ ion and nitroxide spin labels on the RNA. It was found to be preferably situated in the minor groove of the double strand region close to the HP92 loop.

Dynamic Nuclear Polarization (DNP) experiments on solid dielectrics can be described in terms of the Solid Effect (SE) and Cross Effect (CE) mechanisms. These mechanisms are best understood by following the spin dynamics in electron-nuclear and electron-electron-nuclear model systems, respectively. Recently it was shown that the frequency swept DNP enhancement profiles can be reconstructed by combining basic SE and CE DNP spectra. However, this analysis did not take into account the role of the electron spectral diffusion (eSD), which can result in a dramatic loss of electron polarization along the EPR line. In this paper we extend the analysis of DNP spectra by including the influence of the eSD process on the enhancement profiles. We show for an electron-electron-nuclear model system that the change in nuclear polarization can be caused by direct MW irradiation on the CE electron transitions, resulting in a direct CE (dCE) enhancement, or by the influence of the eSD process on the spin system, resulting in nuclear enhancements via a process we term the indirect CE (iCE). We next derive the dependence of the basic SE, dCE, and iCE DNP spectra on the electron polarization distribution along the EPR line and on the MW irradiation frequency. The electron polarization can be obtained from ELDOR experiments, using a recent model which describes its temporal evolution in real samples. Finally, DNP and ELDOR spectra, recorded for a 40 mM TEMPOL sample at 10-40 K, are analyzed. It is shown that the iCE is the major mechanism responsible for the bulk nuclear enhancement at all temperatures.

Quantitative cysteine-independent ligation of a Gd3+ tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd3+-Gd3+ distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd3+ ion relative to the protein can be predicted with high accuracy.

The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+-based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multi-spin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 -> Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

Methods for measuring nanometer scale distances between specific sites in biomolecules (proteins and nucleic acids) and their complexes are essential for describing and analyzing their structure and function. In the last decade pulse EPR techniques were proven very effective for measuring distances between two spin labels attached to a biomolecule. The most commonly used spin labels for such measurements are nitroxide stable radicals. Recently, a new family of spin labels, based on Gd3+ chelates, has been introduced to overcome some of the limitations of using nitroxides, particularly at high magnetic fields, which are attractive due to the increased sensitivity they offer. The benefits that such S = 7/2 spin labels offer for frequencies of 30 GHz and higher, particularly at 95 GHz, include (1) high sensitivity, only similar to 0.15 nmol of doubly labeled biomolecule is needed, (2) the lack of orientation selection, which allows straightforward data analysis. Gd3+-Gd3+ DEER (double electron-electron resonance) distance measurements on labeled peptides, proteins and DNA have already been demonstrated and the results show that they are very promising in terms of sensitivity. In this Perspective we review these new developments. We briefly introduce the characteristics of the DEER experiment on a pair of S = 1/2 spins and characterize the EPR spectroscopic properties of Gd3+ ions. We then introduce some of the tags employed to attach Gd3+ to biomolecules and provide a few experimental examples of Gd3+-Gd3+ DEER measurements. This is followed by a discussion of the parameters that affect the sensitivity of such DEER measurements. Since an important term in the spin Hamiltonian of Gd3+ is the zero-field splitting (ZFS), its effect on the DEER modulation frequencies must be considered and this is discussed next. Finally, another recently reported approach for using Gd3+ in distance measurements will be presented: the use of Gd3+-nitroxide pairs.

The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of alpha- and beta-chains, which are responsible for antigen recognition. Within the transmembrane domain of the a-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phos-phatidylcholine/cholesterol (9: 1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCR alpha, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

The C-13 solid state Dynamic Nuclear Polarization (DNP) mechanism using trityl radicals (OX63) as polarizers was investigated in the temperature range of 10-60 K. The solutions used were 6 M C-13 urea in DMSO/H2O (50% v/v) with 15 mM and 30 mM OX63. The measurements were carried out at similar to 3.5 T, which corresponds to Larmor frequencies of 95 GHz and 36 MHz for the OX63 and the C-13 nuclei, respectively. Measurements of the C-13 signal intensity as a function of the microwave (MW) irradiation frequency yielded C-13 DNP spectra with temperature dependent lineshapes for both samples. The maximum enhancement for the 30 mM sample was reached at 40 K, while that of the 15 mM sample at 20-30 K. Furthermore, the lineshapes observed showed that both the cross effect (CE) and the solid effect (SE) DNP mechanisms are active in this temperature range and that their relative contribution is temperature dependent. Simulations of the spectra with the relative contributions of the CE and SE mechanisms as a fit parameter revealed that for both samples the CE contribution decreases with decreasing temperature while the SE contribution increases. In addition, for the 15 mM sample the contributions of the two mechanisms are comparable from 20 K to 60 K while for the 30 mM the CE dominates in this range, as expected from the higher concentration. The steep decrease of the CE contribution towards low temperatures is however unexpected. The temperature dependence of the OX63 longitudinal relaxation, DNP buildup times and C-13 spin lattice relaxation times did not reveal any obvious correlation with the DNP temperature dependence. A similar behavior of the CE and SE mechanism was observed for H-1 DNP with the nitroxide radical TEMPOL as a polarizer. This suggests that this effect is a general phenomenon involving a temperature dependent competition between the CE and SE mechanisms, the source of which is, however, still unknown. (C) 2013 Elsevier Inc. All rights reserved.

The organization and orientation of membrane-inserted helices is important for better understanding the mode of action of membrane-active peptides and of protein-membrane interactions. Here we report on the application of ESEEM (electron spin-echo envelope modulation) and DEER (double electron-electron resonance) techniques to probe the orientation and oligomeric state of an a-helical trans-membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin-labeled WALP23 and H-2 nuclei of either the solvent (D2O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of H-2 nuclei in the hydrophilic region of the membrane and the density of the H-2 nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin-labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N-terminus is more buried than the C-terminus. In addition, the ESEEM data fitting yielded a H-2 layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented.

Anionic surfactant-templated mesoporous silicas (AMS) are synthesized with a co-structure directing agent (CSDA) that interacts with both the organic and inorganic components of the system. The AMS materials structure is controlled by pH. We investigated the formation of AMS cubic and hexagonal phases, prepared under the same conditions, except pH, by EPR spectroscopic measurements. We used silica-like and surfactant-like spin probes added to the reaction mixtures in minute amounts. Through the spin probes we resolved the specific interactions of the CSDA (N-trimethoxylsilylpropyl-N,N,N-trimethyl ammonium chloride (TMAPS)) with the surfactant (sodium myristate (C14AS)) and the polymerizing silica. We observed for both phases a fast formation of a mesophase upon addition of the silica precursor (TEOS, tetraethoxysilane) and the CSDA into the surfactant solution, attributed to the strong attraction between the CSDA and the anionic surfactant. This is then followed by a slow condensation of the silica. Electron spin echo envelope modulation (ESEEM) spectra of both spin probes in the as-synthesized materials indicated the presence of two types of CSDA molecules; one interacting with the surfactant and the other with the silica wall. Continuous wave EPR spectra showed different spin probe motilities in the two as-synthesized materials that indicated that the relative populations of the two CSDA types are different in the two phases. We attribute this difference to the pH differences in the reaction mixtures. A soft extraction of the surfactant from the pores did not alter the structure of the final materials, but it abolished the observed molecular level differences between them. The extraction allowed the pending ammonium groups to acquire a high degree of freedom and accessibility to water molecules. (C) 2012 Elsevier Inc. All rights reserved.

In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc. 2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and S-33 and O-17 labeling. Here we report new results for species 1 of R160Q based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K-a- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of O-17 signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters.

The formation mechanism of the cubic mesoporous carbon, FDU-16, synthesized by evaporation-induced self-assembly (EISA) was investigated at the molecular level by electron paramagnetic resonance (EPR) spectroscopic techniques. This material is synthesized using F127 pluronic block copolymer [poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO106-PPO70-PEO106)] as a structure-directing agent (template) and phenolic resol as a carbon precursor. Using two spin probes derived from pluronics with PEO and PPO chains of different lengths that are designed to sense different regions of the system, we followed the evaporation and thermopolymerization stages of the synthesis in situ. To make such studies possible, we have used a polyurethane foam support, placed in the EPR tube, which allows for the efficient solvent evaporation as required for EISA. We focused on the evolution of the dynamics of the template and its interactions with the resol during the reaction. We observed that during the evaporation stage the resol is distributed throughout the entire PEO blocks, all the way to the PPO-PEO interface, interacting with them via H-bonds, thus hindering the local motion of the PEO chains. At the end of this stage there is no polarity gradient along the PEO blocks, as found for traditional F127 micelles in water or during the synthesis of silica materials, and the mesostructure is not well-defined. A polarity and a resol gradient developed during the thermopolymerization stage where the polymerizing resol is driven out to the outer region of the PEO corona. This produces a corona of resin-pluronic composite and a resol-free PPO core with high mobility of the PEO segments close to the PPO-PEO interface and restricted mobility in the composite corona. During this stage the final structure sets in.

Studies of membrane peptide interactions at the molecular level are important for understanding essential processes such as membrane disruption or fusion by membrane active peptides. In a previous study, we combined several electron paramagnetic resonance (EPR) techniques, particularly continuous wave (CW) EPR, electron spin echo envelope modulation (ESEEM), and double electron-electron resonance (DEER) with Monte Carlo (MC) simulations to probe the conformation, insertion depth, and orientation with respect to the membrane of the membrane active peptide melittin. Here, we combined these EPR techniques with cryogenic transmission electron microscopy (cryo-TEM) to examine the effect of the peptide/phospholipid (P/PL) molar ratio, in the range of 1:400 to 1:25, on the membrane shape, lipids packing, and peptide orientation and penetration. Large unilamellar vesicles (LUVs) of DPPC/PG (7:3 dipalmitoylphosphatidylcholine/egg phosphatidylglycerol) were used as model membranes. Spin-labeled peptides were used to probe the peptide behavior whereas spin-labeled phspholipids were used to examine the membrane properties. The cryo-TEM results showed that melittin causes vesicle rupture and fusion into new vesicles with ill-defined structures. This new state was investigated by the EPR methods. In terms of the peptide, CW EPR showed decreased mobility, and ESEEM revealed increased insertion depth as the P/PL ratio was raised. DEER measurements did not reveal specific aggregates of melittin, thus excluding the presence of stable, well-defined pore structures. In terms of membrane properties, the CW EPR reported reduced mobility in both polar head and alkyl chain regions with increasing P/PL. ESEEM measurements showed that, as the P/PL ratio increased, a small increase in water content in the PL headgroup region took place and no change was observed in the alkyl chains part close to the hydrophilic region. In terms of lipid local density, opposite behavior was observed for the

Exchange-coupled spin triads nitroxide-copper(II)-nitroxide are the key building blocks of molecular magnets Cu(hfac)(2)L-R. These compounds exhibit thermally induced structural rearrangements and spin transitions, where the exchange interaction between spins of copper(II) ion and nitroxide radicals changes typically by 1 order of magnitude. We have shown previously that electron paramagnetic resonance (EPR) spectroscopy is sensitive to the observed magnetic anomalies and provides information on both inter- and intracluster exchange interactions. The value of intracluster exchange interaction is temperature-dependent (J(T)), that can be accessed by monitoring the effective g-factor of the spin triad as a function of temperature (g(eff)(T)). This paper describes approaches for studying the g(eff)(T) and J(T) dependences and establishes correlations between them. The experimentally obtained g(eff)(T) dependences are interpreted using three different models for the mechanism of structural rearrangements on the molecular level leading to different meanings of the J(T) function. The contributions from these mechanisms and their manifestations in X-ray, magnetic susceptibility and EPR data are discussed.

Nitroxide spin probe electron paramagnetic resonance (EPR) has proven to be a very successful method to probe local polarity and solvent hydrogen bonding properties at the molecular level. The g(xx) and the N-14 hyperfine A(zz) principal values are the EPR parameters of the nitroxide spin probe that are sensitive to these properties and are therefore monitored experimentally. Recently, the N-14 quadrupole interaction of nitroxides has been shown to be also highly sensitive to polarity and H-bonding (A. Savitsky et al., J. Phys. Chem. B 112 (2008) 9079). High-field electron spin echo envelope modulation (ESEEM) was used successfully to determine the P-xx and P-yy principal components of the N-14 quadrupole tensor. The P-zz value was calculated from the traceless character of the quadrupole tensor. We introduce here high-field (W-band, 95 GHz, 3.5 T) electron-electron double resonance (ELDOR)-detected NMR as a method to obtain the N-14 P-zz value directly, together with A(zz). This is complemented by W-band hyperfine sublevel correlation (HYSCORE) measurements carried out along the g(xx) direction to determine the principal P-xx and P-yy components. Through measurements of TEMPOL dissolved in solvents of different polarities, we show that A(zz) increases, while vertical bar P-zz vertical bar decreases with polarity, as predicted by Savitsky et al. (C) 2011 Elsevier Inc. All rights reserved.

cd(1) nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO), It contains a specialized d(1)-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d(1)-heme to avoid enzyme inhibition by the formation of ferrous nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d(1)-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzola, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d(1)-heme NO complex from Pseudomonas aeruginosa cd(1) NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr(10), His(327), and His(369)), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (END OR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the END OR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d(1)-heme complex of cd, NIR forms H-bonds with Tyr(10) and His(369), whereas the second conserved histidine, His(327), appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr(10) is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was sh

We present high field DEER (double electron-electron resonance) distance measurements using Gd3+ (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd3+ spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd3+ was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd3+ labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd3+ labeled peptide we explored the effect of the solution molar ratio of Gd3+ and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd3+]/[Tag] ratio is crucial because excess Gd3+ masked the DEER effect and too little Gd3+ resulted in the formation of Gd3+-tag2 complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd3+ concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd3+ ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.

In this work, we continue to explore Gd(III) as a possible spin label for high-field Double Electron-Electron Resonance (DEER) based distance measurements in biological molecules with flexible geometry. For this purpose, a bis-Gd(III) complex with a flexible "bridge" was used as a model. The distances in the model were expected to be distributed in the range of 5-26 angstrom. allowing us to probe the shortest limits of accessible distances which were found to be as small as 13 angstrom. The upper distance limit for these labels was also evaluated and was found to be about 60 angstrom. Various pulse duration setups can result in apparent differences in the distribution function derived from DEER kinetics due to short distance limit variations. The advantages, such as the ability to perform measurements at cryogenic temperatures and high repetition rates simultaneously, the use of very short pumping and observation pulses without mutual interference, the lack of orientational selectivity, as well as the shortcomings, such as the limited mw operational frequency range and intrinsically smaller amplitude of oscillation related to dipolar interaction as compared with nitroxide spin labels are discussed. Most probably the use of nitroxide and Gd-based labels for distance measurements will be complementary depending on the particulars of the problem and the availability of instrumentation. (C) 2010 Elsevier Inc. All rights reserved.

Self-assembly (SA) of amphiphilic block copolymers (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)) was investigated in dispersions of multi-walled carbon nanotubes (MWNT) as a function of temperature using Spin probe electron paramagnetic resonance (EPR) spectroscopy. Nitroxide-labeled Pluronic with a short poly(ethylene oxide) block, L62-NO, and a small molecular probe, 4-hydroxy-TEMPO-benzoate, 4HTB, were used for probing the local dynamic and polarity of the polymer chains in the presence of the nanostructures. It was found that MWNT modify the temperature, and the dynamic behavior of polymer SA and comparison between the MWNT and single-walled nanotube (SWNT) showed that the structure and dynamical behavior of the nanostructure-polymer hybrids formed depend on the size matching between the diameter of the native micelles and the additives. While SWNT induced the formation of hybrid polymer-SWNT micelles, MWNT (witha diameter of 20-40 nm) induced the assembly of polymer aggregates at the Surface of the MWNT.

A simple setup for rapid freeze-quench electron paramagnetic resonance (EPR) at W-band is described. It is based on a BioLogic commercial apparatus and a modified sample collection appropriate for W-band capillaries. The standard reaction of myoglobin with azide, which converts high-spin Fe(III) to low-spin Fe(III), used for calibration of rapid freeze-quench X-band EPR is very inconvenient for high-field measurements. Here we propose a different simple calibration reaction for W-band: the reduction of a nitroxide free radical with sodium dithionite using Mn2+ as an internal standard. Using this calibration reaction we determined the dead time of our system to be less than 10 ms.

We studied the structural evolution during the formation of large-pore cubic laid silica-based mesoporous materials, synthesized with Pluroinc P123 and butanol as structure directing agents. We-used cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM). Typically a silica precursor is added to an acid-catalyzed solution of Pluronic P123 and butanol. The latter serves as a cosolute, which can be added either at the beginning of the reaction, or after precipitation and the formation of a hexagonal phase. In this study we focused on the structural evolution from the hexagonal phase to the final cubic phase in the two different reactions. The same structural evolution with different kinetics was detected for both reactions. Cryo-HRSEM and FFR-TEM images revealed that from the hexagonal phase a perforated layer (PL) phase is formed, which later evolves into a bicontinuous structure. The final cubic phase forms within the layers, maintaining their orientation. We suggest a formation mechanism involving cylinder merging for the hexagonal to PL transition. Upon additional polymerization of the silica, the PL phase relaxes into the stable la (3) over bard cubic phase. Another minor mechanism detected involves the direct transition between the hexagonal to the final cubic phase through cylinder branching.

W-band (95 GHz) HYSCORE and pulse ENDOR are used to characterize the nitrosyl d(1) heme complex (d(1)NO) of cd(1) nitrite reductase from Pseudomonas aeruginosa in the wild type and the Y10F mutant. The spectra and the derived N-14 hyperfine and quadrupole interactions were found to be the same for wt and Y10F. This suggests that Tyr10 does not influence the NO ligand orientation in the reduced state in solution. This study is the first application of HYSCORE at high fields and shows its potential for characterizing low gamma nuclei with large hyperfine couplings.

High resolution pulse EPR techniques applied to half integer high spin systems, such as Mn(2+) (S = 5/2), usually focus only on the central vertical bar -1/2 > -> vertical bar 1/2 > transition. The reason is that at high fields, where the zero field splitting is considerably smaller than the Zeeman interaction, the spectrum of this transition is intense and narrow. However, because the experiments are carried out at low temperatures, the low lying levels are heavily populated and the signal of the central transition is nevertheless diminished. This, in turn affects the sensitivity of the pulse EPR technique applied. A transfer of populations from the lower lying levels, which for Mn(2+) are the vertical bar-3/2 > and vertical bar-5/2 > levels, to the vertical bar-1/2 > level will therefore increase the sensitivity. Here we describe such an experiment, where a rapid magnetic field sweep over the vertical bar-3/2 > -> vertical bar-1/2 > sub- spectrum is carried out, concomitantly with a low power microwave (mw) irradiation, which results in population inversion. After this sweep any pulsed EPR sequence can be applied to the central transition that now has a population difference that deviates from the equilibrium value. The feasibility of the experiment is demonstrated at W-band (95 GHz) on Mn(2+) doped in MgO for echo-detected EPR measurements and the dependence of the signal enhancement on the rate and range of the magnetic field sweep and on the mw power is described. The results are then accounted for theoretically by considering a simple fictitious spin 1/2 system. In addition, preliminary enhanced (55)Mn pulse ENDOR electron nuclear double resonance (ENDOR) spectra are presented.

(100-x) mol % B(2)O(3) x mol % Me(2)O (Me=Li,Na,K) glasses, exposed to gamma-(60)Co irradiation to produce paramagnetic states, were characterized by W-band (95 GHz) pulse electron-nuclear double resonance (ENDOR) spectroscopy in order to characterize local structures occurring in the range of compositions between x=16 and x=25 at which the "boron oxide" anomaly occurs. The high resolution of nuclear frequencies allowed resolving the (7)Li and (11)B ENDOR lines. In the samples with x=16 and x=20 glasses, (11)B hyperfine couplings of 16, 24, and 36 MHz were observed and attributed to the tetraborate, triborate, and boron oxygen hole center (BOHC) structures, respectively. The x=25 samples showed hyperfine couplings of 15 MHz for the tetraborate and 36 MHz for BOHC. Density functional theory (DFT) calculations predicted for these structures negative hyperfine couplings, which were confirmed by W-band ENDOR. This suggests that a spin polarization mechanism accounts for the negative hyperfine structure splitting. (C) 2008 American Institute of Physics. [DOI: 10.1063/1.2991171]

The self-assembly (SA) of amphiphilic block copolymers (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)) was investigated in dispersions of single-walled and multiwalled carbon nanotubes (SWNT and MWNT, respectively)as a function of temperature. Differential scanning calorimetry (DSC) was used for characterization of the thermal behavior of the combined polymers-nanostructures system, and spin-probe electron paramagnetic resonance (EPR) was employed for probing the local dynamic and polarity of the polymer chains in the presence of nanostructures. It was found that SWNT and MWNT modify the temperature, enthalpy, and dynamic behavior of polymer SA. In particular, SWNT were found to increase the cooperativity of aggregating chains and dominate aggregate dynamics. MWNT reduced the cooperativity, while colloidal carbon black additives, studied for comparison, did not show similar effects. The experimental observations are consistent with the suggestion that dimensional matching between the characteristic radius of the solvated polymer chains and the dimensions of additives dominate polymer SA in the hybrid system.

The self-assembly of Pluronic block copolymers in dispersions of single-wall carbon nanotubes (SWNT) was investigated by spin probe electron paramagnetic resonance (EPR) spectroscopy. Nitroxide spin labeled block copolymers derived from Pluronic L62 and P123 were introduced in minute amounts into the dispersions. X-band EPR spectra of the SWNT dispersions and of native polymer solutions were measured as a function of temperature. All spectra, below and above the critical micelle temperature (CMT), were characteristic of the fast limit motional regime. The temperature dependence of the N-14 isotropic hyperfine coupling, a(iso), and the rotational correlation time, tau(c), were determined. It was observed that, below the CMT, EPR does not distinguish between chains adsorbed on SWNT and free chains. Above CMT, substantial differences were observed: in the native solution, the Pluronics spin labels experience only one environment, S-m, assigned to spin labels in the corona of the Pluronic micelle, whereas in the SWNT dispersions, in addition to S-m, a second population of nonaggregated, individual chains, S-i, is observed. The relative amounts of S-m and S-i were found to depend on the relative concentrations of the Pluronic and SWNT. Furthermore, the aggregates formed in the SWNT dispersions do not show the typical increase in chain-end mobility as a function of temperature, observed in the post-CMT regime of the native Pluronic solutions. This suggests a larger dynamical coupling among aggregated chains in the presence of the SWNT as compared to the native micelles. The overall findings are consistent with the formation of a new type of aggregates, composed of a SWNT-polyrner hybrid.

In the overwhelming majority of the exchange-coupled clusters investigated in field of molecular magnetism, the exchange interaction is constant on temperature. ''Breathing'' crystals of composition Cu(hfac)(2)L-R undergo temperature-induced reversible structural rearrangements accompanied by significant changes of the effective magnetic moment. Using high-field (W-band) EPR, we provide a solid proof of drastic temperature dependence of exchange interaction J(T) in these compounds that originates from temperature dependence of inter-spin distances. Strong dependence J(T) revealed by EPR makes Cu(hfac)(2)L-R breathing crystals interesting and promising systems in the research toward creation of molecular-magnetic switches and related spin devices.

Davies electron-nuclear double resonance spectra can exhibit strong asymmetries for long mixing times, short repetition times, and large thermal polarizations. These asymmetries can be used to determine nuclear relaxation rates in paramagnetic systems. Measurements of frozen solutions of copper(L-histidine)(2) reveal a strong field dependence of the relaxation rates of the protons in the histidine ligand, increasing from low (g(parallel to)) to high (g(perpendicular to)) field. It is shown that this can be attributed to a concentration-dependent T-1e-driven relaxation process involving strongly mixed states of three spins: the histidine proton, the Cu(II) electron spin of the same complex, and another distant electron spin with a resonance frequency differing from the spectrometer frequency approximately by the proton Larmor frequency. The protons relax more efficiently in the g(perpendicular to) region, since the number of distant electrons able to participate in this relaxation mechanism is higher than in the g(parallel to) region. Analytical expressions for the associated nuclear polarization decay rate T-een(-1) are developed and Monte Carlo simulations are carried out, reproducing both the field and the concentration dependences of the nuclear relaxation. (C) 2007 American Institute of Physics.

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. Mn-55 electron-nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, similar to 3.5 T) to determine the Mn-55 nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the Mn-55 ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e(2)Qq/h, are significantly different; 10.7 +/- 0.6 MHz for Mn-B(2+.) The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for Mn-A(2+) and -263.5 MHz for Mn-B(2+.) The principle z-axis for Mn-A(2+) is not aligned with any of the Mn-ligand directions, but is 25% off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of Mn-B(2+) the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90 degrees with respect to Mn-A. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at l

Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 angstrom, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and H-1 ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the H-1 ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site I

An EPR spectrum of as synthesized [G.A. Tsigdinos, C.J. Hallada, Inorg. Chem. 7 (1968) 437-441], orange colored, H5PV2Mo10O40 polyoxometalate showed the presence of a reduced vanadium(IV) addenda atom. Surprisingly, further P-31 ENDOR (electron-nuclear double resonance) measurements indicated the absence of a phosphorous heteroatom leading to the suggestion that (H5VVMo11O40)-V-V-Mo-IV exists as a previously unrecognized impurity in the typically synthesized H5PV2Mo10O40 compound. (H5/4PVO4VMo11O36)-O-V-Mo-IV/V was then synthesized in low yield (0.8 mol%) by omitting the addition of phosphate in a typical H5PV2Mo10O40 preparation. The molecular formulation and structure was supported by X-ray crystallography, infrared and mass spectrometry. Further use of EPR/ENDOR/ESEEM (electron-spin echo envelope modulation) allowed the formulation of [(VVMo11O40)-V-V-Mo-IV](5-) as [(VO4VMo11O36)-O-V-Mo-IV](5-). Accordingly, the polyoxometalate has a VO43- heteroatom core with 11 molybdenum addenda and one VO2+ moiety at the polyoxometalate surface. The redox potential and the catalytic activity of the new vanadomolybdate polyoxometalate compound were essentially identical to the often-studied H5PV2Mo10O40 polyoxometalate isomeric mixture.

The evolution of the solution microstructures during the formation of the hexagonal mesoporous material SBA-15 was studied by direct imaging and freeze-fracture replication cryo-TEM, A reaction mixture was sampled at different times after the addition of tetramethoxyorthosilane (TMOS) to an acidic solution of Pluronic P123 held at 50 degrees C. Solution microstructures were detected by direct imaging cryo-TEM in the time window of 6.5-40 min after the addition of the TMOS (t = 0). The micrographs revealed that the initial spheroidal micelles evolve into threadlike micelles, which become longer and straighter with time. Then bundles with the dimensions similar to those found in the final material appeared, although there was no sign of a hexagonal arrangement up to 40 min. Due to the appearance of a precipitate at 40 min the sample became too viscous, preventing clear observation of its content. To observe the structures present after 40 min, freeze-fracture replication was carried out as well. Such samples were collected also at 22 min and showed the presence of threadlike micelles in agreement with the direct imaging cryo-TEM micrographs. The 2 h samples showed some areas of hexagonal ordered structures, which become very clear at 2 h 50 min. The cryo-TEM measurements were carried out under the same reaction conditions used in earlier in situ EPR experiments, thus allowing us to correlate molecular level events with the microstructure shape evolutions. This showed that the elongation of the micelles is a consequence of a reduction of the polarity and the water content within the micelles due to silicate adsorption and polymerization. Similar experiments were carried out also on SBA-15 prepared with HCI and TMOS at 35 degrees C. The appearance of threadlike micelles, followed by clustering of the TLMs, was observed under these conditions as well, but the reaction rate was faster. This suggests that the observed mechanism for the formation of SBA-15 is general.

Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration pro. le of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26- mer bee venom melittin and a de novo designed 15-mer D, L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine ( DPPC) and phosphatidylglycerol ( PG), (DPPC/PG (7:3w/w)), egg phosphatidylcholine ( PC) and PG (PC/PG (7:3w/w)), and phosphatidylethanolamine ( PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2 H nuclei was determined and compared with a series of controls that produced a reference ''ruler''. Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it ''flatter'' and increasing the penetration depth into the hydrophobic region.

The properties of the Mn2+ site in the protein concanavalin A were investigated by single crystal W-band EPR/ENDOR (electron-nuclear double resonance) measurements. Initially, room temperature EPR measurements were carried out, one type of Mn2+ was identified and its zero-field splitting (ZFS) tensor was determined. In contrast, low temperature EPR measurements showed that two chemically inequivalent Mn2+ are present, Mn-A(2+) and Mn-B(2+), differing in their ZFS tensors. Variable temperature measurements revealed a two-site exchange between the two types. Although the dynamic process has been characterized by its rate and activation energy, just from the EPR measurements it was not possible to assign it to a specific residue. H-1 ENDOR measurements of the water and imidazole protons, which are the main contributors to the ENDOR spectra, showed only one type of signals, namely, they were not sensitive to the differences between Mn-A(2+) and Mn-B(2+). Mn-55 ENDOR spectra, which are dominated by the Mn-55 isotropic hyperfine, a(iso), and the nuclear quadrupole interaction did sense the differences. Analysis of the spectra recorded with the magnetic field along the crystallographic axes showed that the two have the same a(iso), but different quadrupole tensors. Copyright (c) 2005 John Wiley & Sons, Ltd.

A versatile control software for pulse EPR spectrometers is introduced. Common and task-specific problems are discussed and their solutions are described. The software provides the full spectrum of possibilities needed to perform arbitrary multidimensional pulse experiments. It allows for an easy interfacing of commonly used hardware components and enables straightforward modifications of the spectrometer. Good performance, configurability, and a number of unique features turn this software into an excellent tool for the operation of modern EPR spectrometers and upgrading old ones. (c) 2005 Wiley Periodicals, Inc.

The properties of the silica layer during the formation of the mesoporous material MCM-41 were investigated by electron paramagnetic resonance (EPR) experiments carried out on a specifically designed, organo(trialkoxy)silane spin probe, SL1SiEt. Minute amounts of the spin probe were co-condensed with the silica source, tetraethyl orthosilicate (TEOS), in the synthesis of MCM-41 with cetyltrimethylammonium bromide (CTAB) under basic conditions. The mobility and location of the spin probe were followed in the CTAB micellar solution before the reaction, in the reaction mixture and in the final ordered material. It was found that the EPR spectra of hydrolyzed SL1SiEt throughout the room temperature part of the reaction are characteristic of a fast tumbling species, indicating that the silica is highly fluid prior to drying. After filtering, a slow motion type spectrum was observed, showing that the spin-label experiences considerable motional hindrance. The liquidlike behavior could be restored upon stirring the material in water. When the reaction is performed with a hydrothermal stage, the spectrum of SL1SiEt in the final product is the same as that of the room temperature synthesized material, but the addition of water did not restore the high mobility, due to a higher degree of silica cross-linking. The location of SL1SiEt throughout the formation process was obtained from electron spin-echo envelope modulation (ESEEM) measurements on MCM-41 prepared with CTAB deuterated either at the N-methyl or the alpha position and in a reaction carried out in D(2)O. Comparing the deuterium modulation depth, k((2)H), induced by CTAB-alpha-d2, CTAB-d(9), or D(2)O in CTAB micellar solutions of a number of reference spin probes with those of SL1SiEt revealed that the hydrolyzed SL1SiEt is located near the polar heads of the surfactant in the absence of base and TEOS. This supports the postulation of charge matching at the interface as a driving force for the formation of the mes

In this work we have studied pulsed O-17 electron nuclear double resonance (ENDOR) spectra of the Gd3+ aquo ion and the magnetic resonance imaging (MRI) contrast agent MS-325 in an O-17-enriched frozen glassy water/methanol solution. The isotropic hyperfine interaction (hfi) constant of the water ligand 17(O) was found to be about 0.75 MHz, which corresponds to a spin density delocalized to the ligand of rho(O) approximate to -4 x 10(-3). The analysis of the anisotropic hfi constant (0.69 +/- 0.05 MHz) yields Gd-O distances of about 2.4-2.5 (A) over circle. Simultaneous analysis of these distances and the Gd-H distances found earlier allows one to elucidate the details of the Gd-OH2 coordination geometry.

The two-dimensional (2D) TRIPLE experiment provides correlations between electron-nuclear double resonance (ENDOR) frequencies that belong to the same electron-spin manifold, M-S, and therefore allows to assign ENDOR lines to their specific paramagnetic centers and M-S manifolds. This, in turn, also provides the relative signs of the hyperfine couplings. So far this experiment has been applied only to single crystals, where the cross-peaks in the 2D spectrum are well resolved with regular shapes. Here we introduce the application of the 2D TRIPLE experiment to orientationally disordered systems, where it can resolve overlapping powder patterns. Moreover, analysis of the shape of the cross-peaks shows that it is highly dependent on the relative orientation of the hyperfine tensors of the two nuclei contributing to this particular peak. This is done initially through a series of Simulations and then demonstrated experimentally at a high field (W-band, 95 GHz). The first example concerned the 1 H hyperfine tensors of the stable radical alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) immobilized in a polystyrene matrix. Then, the experiment was applied to a more complex system, a frozen solution of Cu(II)-bis(2,2':6',2" terpyridine) complex. There, the 2D TRIPLE experiment was combined with the variable mixing time (VMT) ENDOR experiment, which determined the absolute sign of the hyperfine couplings involved, and orientation selective ENDOR experiments. Analysis of the three experiments gave the hyperfine tensors of a few coupled protons. (C) 2004 Elsevier Inc. All rights reserved.

Two current frontiers in EPR research are high-field (nu(0) > 70 GHz, B-0 > 2.5 T) electron paramagnetic resonance (EPR) and high-field electron-nuclear double resonance (ENDOR). This review focuses on recent advances in high-field ENDOR and its applications to the study of proteins containing native paramagnetic sites. It concentrates on two aspects; the first concerns the determination of the location of protons and is related to the site geometry, and the second focuses on the spin density distribution within the site, which is inherent to the electronic structure. Both spin density and proton locations can be derived from ligand hyperfine couplings determined by ENDOR measurements. A brief description of the experimental methods is presented along with a discussion of the advantages and disadvantages of high-field ENDOR compared with conventional X-band (similar to9.5 GHz) experiments. Specific examples of both protein single crystals and frozen solutions are then presented. These include the determination of the coordinates of water ligand protons in the Mn(II) site of concanavalin A, the detection of hydrogen bonds in a quinone radical in the bacterial photosynthetic reaction center as well as in the tyrosyl radical in ribonuclease reductase, and the study of the spin distribution in copper proteins. The copper proteins discussed are the type I copper of azurin and the binuclear Cu-A center in a number of proteins. The last part of the review presents a brief discussion of the interpretation of hyperfine couplings using quantum chemical calculations, primarily density functional theory (DFT) methods. Such methods are becoming an integral part of the data analysis tools, as they can facilitate signal assignment and provide the ultimate relation between the experimental hyperfine couplings and the electronic wave function.

Two approaches for improving the signal-to-noise ratio (S/N) of W-band pulsed electron-nuclear double resonance (ENDOR) spectra are presented. One eliminates base-line problems while the other enhances the ENDOR effect. High field ENDOR spectra measured at low temperatures often suffer from highly distorted base-lines due to the heating effect of the RF pulses that causes some detuning of the cavity and therefore leads to a reduction in the echo intensity. This is a severe problem because it often masks broad and weak ENDOR signals. We show that it can be eliminated by recording the ENDOR spectrum in a random, rather than the standard sequential variation of the RF frequency. The S/N of the ENDOR spectrum can be significantly enhanced by the application of the pulse analog of the continuous wave (CW) special TRIPLE experiment. While this experiment is not applicable in the solid state at conventional X-band frequencies, at W-band it is most efficient. We demonstrate the efficiency of the special TRIPLE Davies and Mims experiments on single crystals and orientationally disordered systems. (C) 2003 Elsevier Inc. All rights reserved.

EPR spectroscopy at 95 GHz was used to characterize the dynamics at the Mn2+ binding site in single crystals of the saccharide-binding protein concanavalin A. The zero-field splitting (ZFS) tensor of the Mn2+ was determined from rotation patterns in the a-c and a-b crystallographic planes, acquired at room temperature and 4.5 K. The analysis of the rotation patterns showed that while at room temperature there is only one type of Mn2+ site, at low temperatures two types of Mn2+ sites, not related by any symmetry, are distinguished. The sites differ in the ZFS parameters D and E and in the orientation of the ZFS tensor with respect to the crystallographic axes. Temperature-dependent EPR measurements on a crystal oriented with its crystallographic a axis parallel to the magnetic field showed that as the temperature increases, the two well-resolved Mn2+ sextets gradually coalesce into a single sextet at room temperature. The line shape changes are characteristic of a two-site exchange. This was confirmed by simulations which gave rates in the range of 10(7)-10(8) s(-1) for the temperature range of 200-266 K and an activation energy of 23.8 kJ/mol. This dynamic process was attributed to a conformational equilibrium within the Mn2+ binding site which freezes into two conformations at low temperatures.

High-field EPR and pulsed electron-nuclear double resonance (ENDOR) spectroscopies were used to investigate the formation of Mn-AlPO4-11, Mn-AlPO4-5, and Mn-SAPO-5. Samples recovered from reaction mixtures quenched at different times were subjected to EPR, ENDOR and X-ray diffraction (XRD) measurements, and the variations in the (31)p and H-1 hyperfine couplings, which are sensitive probes to the Mn-P interaction and the Mn(II) hydration, respectively, were followed. The intensity of the 1H ENDOR signal decreased with reaction time, showing that the amount of both water ligands and solvent water in the Mn(II) vicinity decreased. A relatively large isotropic P-31 hyperfine coupling (A(iso)((31)p) approximate to 7 MHz), confirming the formation of Mn(II) framework sites, was found in all final products, whereas a smaller A(iso)(P-31), 4-5 MHz, was detected in samples quenched at early stages of the reaction. The latter was assigned to Mn(II) incorporated into a network of disordered aluminophosphate precursors. These precursors are formed prior to the detection of an XRD pattern, and are gradually transformed to the final three-dimensional crystalline structures. The changes in A(iso)(P-31) were attributed to transformations occurring both in the bonding topology and in the coordination sphere of Mn(II), where water ligands are gradually replaced by -O-P linkages. This interpretation was supported by the decrease in the intensity of the H-1 ENDOR signals, and by a series of DFT cluster model optimizations on intermediates of the form [Mn(H2O)x(OP(OH)(3))y](2+), where x + y = 6, 5 or 4, followed by calculations of hyperfine coupling constants. Although the theoretical hyperfine values were overestimated with respect to the experimental ones, a satisfactory correlation was found between the trends within the calculated A(iso)(Mn-55,(31)p), and the experimental trends observed during the molecular sieves formation.

High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating CUA center. These include nitrous oxide reductase (N2OR), the recombinant water-soluble fragment of subunit 11 of Thermus thermlophilus cytochrome c oxidase (COX) ba(3) (M16OT9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azurin (purpAz). The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known. The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type 11 copper. At W-band, the g-L features of this center and Of CUA are well resolved, thus allowing us to obtain a clean CUA ENDOR spectrum. The latter consists of two types of ENDOR signals. The first includes the signals of the four strongly coupled cysteine beta-protons, with isotropic hyperfine couplings, A(iso), in the 7-15 MHz range. The second group consists of weakly coupled protons with a primarily anisotropic character with A(zz)

High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn2+ ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H2O and in D2O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of similar to3.4 T allowed us to assign the ENDOR signals to their respective Ms manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn2+ ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein.

Electron spin resonance, pulsed electron nuclear double resonance (ENDOR) spectroscopy at Wand X-band frequencies, and hyperfine sublevel correlation (HYSCORE) spectroscopy have been employed to determine the location of the V(IV) ions in H4PVMo11O40 heteropolyacid catalysts. In these materials the heteropolyanions have the well-known structure of the Keggin molecule. Interactions of the unpaired electrons of the paramagnetic vanadyl ions (VO2+) with all relevant nuclei (H-1, P-31, and V-51) could be resolved. The complete analysis of the hyperfine coupling tenser for the phosphorus nucleus in the fourth coordination sphere of the V(IV) ion allowed for the first time a detailed structural analysis of the paramagnetic ions in heteropolyacids in hydrated and dehydrated catalysts. The P-31 and H-1 ENDOR results show that V(IV) ions are incorporated as vanadyl pentaaqua complexes [VO(H2O)(5)](2+) in the void space between the heteropolyanions in the hydrated heteropolyacid. For the dehydrated H4PVMo11O40 materials the distance between the V(IV) ion and the central phosphorus atom of the Keggin molecule could be determined with high accuracy on the basis of orientation-selective P-31 ENDOR experiments and HYSCORE spectroscopy. The results give a first direct experimental evidence that the paramagnetic vanadium species are not incorporated at molybdenum sites into the Keggin structure of H4PVMo11O40 and also do not act as bridges between two Keggin units after calcination of the catalyst. The vanadyl species are found to be directly attached to the Keggin molecules. The VO2+ ions are coordinated to four or three outer oxygen atoms from one PVMo11O40-4 heteropolyanion in a trigonal-pyramidal or slightly distorted square-pyramidal coordination geometry, respectively.

W-band (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) spectroscopic techniques were used to determine the hyperfine couplings of different protons of Cu(II)histidine complexes in frozen solutions. The results were then used to obtain the coordination mode of the tridentate histidine molecule and to serve as a reference for Cu(II)-histidine complexation in other, more complex systems. Cu(LI) complexes with L-histidine and DL-histidine-alpha -d,beta -d(2) were prepared in H2O and in D2O and orientation-selective W-band H-1 and H-2 pulsed ENDOR spectra of these complexes were recorded at 4.5 K. These measurements lead ro the unambiguous assignment of the signals of the H-alpha, H-beta, imidazole H-epsilon, and the exchangeable amino, H-am, protons. The N-14 superhyperfine splitting observed in the X-band EPR spectrum and the presence of only one type of H-alpha and H-beta protons in the W-band ENDOR spectra show that the complex is a symmetric bis complex. Its g(parallel to) is along the molecular symmetry axis, perpendicular to the equatorial plane that consists of four coordinated nitrogens in histamine-like coordinations (NNNN). Simulations of orientation-selective ENDOR spectra provided the principal components of the protons' hyperfine interaction and the orientation of their principal axes with respect to gu. From the anisotropic part of the hyperfine interaction of H-alpha and H-beta and applying the point-dipole approximation, a structural model was derived. An unexpectedly large isotropic hyperfine coupling, 10.9 MHz, was found for H-alpha. In contrast, H-alpha of the Cu(II)-1-methyl-histidine complex, where only the amino nitrogen is coordinated, showed a much smaller coupling. Thus, the hyperfine coupling of H-alpha can serve as a signature for a histamine coordination where both the amino and imino nitrogens of the same molecule bind to the Cu(IT), forming a six-membered chelating ring. Unlike H-alpha the hyperfine coupling of H-epsilo

The coordination geometry of zeolite-encapsulated copper(II)-histidine (CuHis) complexes, prepared by ion exchange of the complexes from aqueous solutions into zeolite NaY, was determined by a combination of UV-vis-NIR diffuse reflectance spectroscopy (DRS), X-band EPR, electron-spin-echo envelope modulation (ESEEM), and high field (W-band) pulsed ENDOR techniques. X-band EPR spectroscopy detected two distinct complexes, A and B, which are different from the prevailing Cu(II) bis-His complex in the exchange solution (PH 7.3 with a His:Cu(II) ratio of 5.1). Moreover, the relative amount of complex B was found to increase with increasing Cu(II) concentration. The EPR parameters of complex A are g(perpendicular to) = 2.054, g(parallel to) = 2.31, and A(parallel to) = 15.8 mT, whereas those of complex B are g(perpendicular to) = 2.068, g(parallel to) =2.25, A(parallel to) = 18.3 mT, and A(perpendicular to)((14)N) similar to 1.3 mT. The presence of the (14)N superhyperfine splitting shows that in complex B three nitrogens are coordinated to the Cu(II). Furthermore, DRS exhibits a shift of the d-d absorption band of Cu(II) from 15 200 cm(-1) in complex A to 15 900 cm(-1) in complex B, indicating an increasing ligand field strength in the latter. The coordination of the imino nitrogen of the imidazole group was detected in the two complexes via the ESEEM frequencies Of the remote nitrogen. In contrast, only complex A exhibited (27)Al modulation, which indicates that the Cu(II) binds to zeolite framework oxygens. (2)H and (1)H W-band ENDOR measurements on samples where the exchangeable protons were replaced with (2)H, and using specifically labeled histidine (His-alpha -d-beta -d(2)), lead to the unambiguous determination of the coordination configuration of the two complexes. Complex A is a mono-His complex when both the amino and imino nitrogens are coordinated and the other equatorial ligands are provided by a zeolite oxygen and a water molecule. Complex B is a bis-Hi

The one-dimensional (1D) pulsed TRIPLE resonance experiment, introduced by Mehring et al. (M. Mehring, P. Hofer, and A. Grupp, Ber. Bunseges. Phys. Chem. 91, 1132-1137 (1987)) is a modification of the standard Davies ENDOR experiment where an additional RF pi-pulse is applied during the mixing time. While the first RF pulse is set to one of the ENDOR transitions, the frequency of the second RF pulse is scanned to generate the TRIPLE spectrum. The difference between this spectrum and the ENDOR spectrum yields the difference TRIPLE spectrum, which exhibits only ENDOR lines that belong to the same M-S manifold as the one selected by the first RF pulse. We have extended this experiment in two dimensions (2D) by sweeping the frequencies of both RF pulses. This experiment is particularly useful when the spectrum is congested and consists of signals originating from different paramagnetic centers. The connectivities between the peaks in the 2D spectrum enable a straightforward assignment of the signals to their respective centers and M-S manifolds, thus providing the relative signs of hyperfine couplings. Carrying out the experiment at high fields has the additional advantage that nuclei with different nuclear gyromagnetic ratios are well separated. This is particularly true for protons which appear at significantly higher frequencies than other nuclei. The feasibility and effectiveness of the experiment is demonstrated at W-band (94.9 GHz) on a crystal of Cu2+-doped L-histidine. Homonuclear H-1-H-1, N-14/Cl-35-N-14/Cl-35 and heteronuclear H-1-N-14/Cl-35 2D TRIPLE spectra were measured and from the various connectivities in the 2D map the H-1, N-14 and Cl-35 signals that belong to two different Cu2+ centers were identified and grouped according to their M-S manifolds. (C) 2000 Academic Press.

The incorporation of Mn(II) into the mesoporous material MCM-41, synthesized under acidic renditions with [Si]/[H(+)] in the range of 0.1-0.4 was investigated. Tetraethyl-orthosilicon (TEOS) was used as the silica sourer and cetyltrimethylammonium chloride or bromide (CTAC/CTAB) as the structure-directing agent. The Mn(II) sires in the final product were characterized by high-field (W-band, 95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) spectroscopies that provided highly resolved EPR spectra and detailed information concerning the Mn(II) coordination sphere. These measurements were complemented with X-band continuous wave (CW) EPR and electron-spin-echo envelope modulation (ESEEM) spectroscopies. hi addition, the bulk properties of the final products were characterized by X-ray diffraction and (29)Si MAS NMR, while in situ X-band CW EPR measurements on reaction mixtures containing the spin probe 5-doxyl stearic acid (5DSA) were carried out to follow the reaction kinetics and the degree of silica condensation. These bulk properties were then correlated with the formation of the different Mn(II) sites. The final product consists of a mixture of two hexagonal phases (d = 38, 43 Angstrom), the relative amounts of which depend on the [Si]/[H(+)] in the starting gel. Two Mn(II) sites, which exhibit unresolved overlapping signal in the X-band EPR spectra, were easily distinguished in the low-temperature field-sweep (FS) echo-detected (ED) W-band spectra due to their significantly different (55)Mn hyperfine couplings. One Mn(II) site, a, is characterized by a hyperfine splitting of 97 G, and the second, b, by 82 G. The relative amount of b increases with increased acidity. Site a is assigned to a hexa-coordinated Mn(II) with water ligands that is anchored to the internal pore surface of the silica either by one coordination site or through hydrogen bond(s). The Mn(II) in site b is in a distorted tetrahedral coordination, located within the first few la

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1 : 1 to 1 : 7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximate to 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximate to 1.97 signal, with similar saturation characteristics was found in the AO : azide (1 : 7) sample. The g

The design and performance of a 95 GHz pulsed W-band EPR/ENDOR spectrometer is described with emphasis on the ENDOR part. Its unique feature is the easy and fast sample exchange at 4.2 It for frozen solution and single crystal samples. In addition, the microwave bridge power output is relatively high (minimum 267 mW), which allows the application of short microwave pulses. This increases the sensitivity in echo experiments because of the broader excitation bandwidth and the possibility of employing short pulse intervals, as long as the dead time does not increase significantly with the power. The spectrometer features two microwave and radiofrequency (0.1-220 MHz, 3 kW pulse power) channels and a 6 T superconducting magnet in a solenoid configuration. The magnet is equipped with cryogenic sweep coils providing a sweep range of +/- 0.4 and +/- 0.2 T for a center field of 0-4 and 4-6 T, respectively. The spectrometer performance is demonstrated on Cu(II) centers in single crystals, a zeolite polycrystalline sample, and a protein frozen solution. (C) 1999 Academic Press.

A versatile high power X-band (8.5-9.5 GHz) pulsed EPR/ENDOR (electron-nuclear double resonance) spectrometer which can generate hundreds of microwave (MW) and rf pulses is described. The pulse programmer is constructed from a word generator with 32 channels and 4 ns resolution, coupled to five digital delay,generators which can produce a total of ten pulses with a resolution better than 1 ns. The spectrometer contains two MW and two rf channels that allow independent variation of the frequency, amplitude, and phase of the MW and rf pulses. The ENDOR probe head is based on a bridged loop gap (BLG) resonator, coupling is achieved via a coupling loop connected to a waveguide, and the rf coil serves as a MW shield as well. The adjustment of the coupling is done by an up/down motion of the of the resonator assembly with respect to the fixed coupling loop. A flexible and user friendly data acquisition program written in C++ (Borland version 4.5), which uses the Windows-95 Multiple Document Interface (MDI) programming model, was developed to run the spectrometer. This program allows easy programming of any pulse sequence with sophisticated phase cycling. The performance of the spectrometer is demonstrated by two experiments. The first is the triple resonance hyperfine-selective (HS) ENDOR experiment carried out on a frozen solution of the copper protein laccase. The second is the two-dimensional hyperfine-ENDOR (HYEND) correlation experiment performed on a single crystal of gamma-irradiated malonic acid. (C) 1998 American Institute of Physics. [S0034-6748(98)02908-6]

Slow exchange processes in the solution of the 1,1'-dihydroxy-2,2',6,6'-tetra-tert-butylbiphenyl cation radical (tBBP(.+)) in concentrated sulfuric acid were investigated by two-dimensional (2D) exchange FT-EPR spectroscopy (EXSY) in the temperature range of 281-310 K. The radical was obtained by dissolving 2,2',6,6'-tetra-tert-butyldiphenoquinone (tBDP) in concentrated sulfuric acid. The EPR spectrum of the radical cation, tBBP(.+), showed that the four aromatic protons and the two hydroxyl protons are magnetically equivalent. The 2D EXSY spectra exhibited cross peaks between hyperfine components with Delta M(I)(a) = +/-1 and Delta M(I)(b) = +/-1, where a and b correspond to the aromatic and hpdroxyl protons, respectively. Based on this selective cross-peak pattern, the change in the nuclear quantum number of the aromatic protons was attributed to proton spin-lattice relaxation. In contrast, the change in the nuclear quantum number of the hydroxyl protons could arise from proton exchange with the solvent and/or nuclear spin-lattice relaxation. Temperature-dependent measurements showed that the intensity of the cross peaks decreased with increasing temperatures, indicating that in the case of the hydroxyl protons the cross peaks are also a consequence of nuclear relaxation and not proton exchange. The nuclear spin-lattice relaxation rates of the two types of protons and the electron spin-lattice relaxation, T-1, were determined from simulations of experimental 2D spectra recorded with different mixing times. The values obtained at 291 K were T-1a(-1) (9 +/- 1) x 10(5) s(-1) and T-1b(-1) = (4 +/- 1) x 10(5) s(-1) and T-1 = (0.83 +/- 0.06) x 10(5) s(-1). Using the nuclear relaxation rate of the aromatic protons and assuming that the nuclear relaxation is dominated by the hyperfine anisotropy mechanism, a correlation time of 0.67 x 10(-9) s was obtained. This value was further used to account for the M(I) dependence of the line width. Similar temperature-dependent 2

Cytochrome P450cam (CP450cam) was studied by pulsed ENDOR and two- and four-pulse ESEEM spectroscopies. Spectra were recorded and simulated at the three principal g-values of the rhombic EPR spectrum. The four-pulse ESEEM experiment gave a direct measure of the anisotropic hyperfine interaction for the protons. Using the point dipole approximation this gives a Fe-H distance of 2.6 Angstrom. The measured anisotropic hyperfine interaction reduced the number of hyperfine interaction parameters required to simulate the ENDOR line shapes. Both the four-pulse ESEEM frequencies and the ENDOR spectra at all three principal g-values could be satisfactorily simulated using two magnetically equivalent protons and a water orientation similar to that obtained in our previous O-17 ESEEM study Thus, the pulsed ENDOR and four-pulse ESEEM results are self-consistent with the O-17 ESEEM data and indicate that the axial ligand is a water molecule rather than an OH- ligand. The isotropic hyperfine value derived from the numerical simulations is in agreement with previous values derived from proton NMR relaxation studies.

Orientation selective and multiple frequency ESEEM experiments on substrate free cytochrome P450cam (CP450cam) with O-17-enriched water are reported. The O-17 ESEEM frequencies were obtained from Fourier transformation of the ratio of ESEEM waveforms of CP450cam with enriched water and CP450cam with nonenriched water. Numerical simulations were carried out to determine the isotropic and the anisotropic hyperfine interactions and the quadrupole interaction of the O-17. From the magnitude (e(2)-qQ/h = 6.6 MHz) and asymmetry (eta = 0.95) of the O-17 quadrupole interaction, we conclude that the distal axial ligand of Fe3+ in the CP450 heme is a water molecule. Moreover, from the orientation of the O-17 quadrupole tenser, the orientation of the water molecule was found to be confined (within +/-10 degrees) with respect to the Fe-N directions within the heme plane. Two possible sets of isotropic and anisotropic components of the O-17 hyperfine interaction, (+/-2.6, +/-0.3 MHz) and (+/-0.4, +/-1.8 MHz), were found to satisfactorily reproduce the experimental results. Both sets indicate very small O-17 hyperfine couplings which is consistent with the unpaired electron residing predominately in the d(yz) orbital of the Fe3+. Ligand field models for each set are presented.

Mesoporous manganosilicate materials Mn-M41S, having hexagonal, cubic and lamellar structures, are synthesized with a low surfactant:Si ratio (0.12:1) at various temperatures and acid/base contents and are characterized by X-ray powder diffraction and Q-band EPR spectroscopy.

Mesoporous manganosilicate molecular sieve, Mn-M41S, having hexagonal (Mn-MCM-41), cubic (Mn-MCM-48), lamellar (Mn-MCM-L) structure, were synthesized in low surfactant/Si ratio (0.12) and characterized by X-ray powder diffraction, transmission electron microscopy (TEM), themogravimetric analysis (TGA) and electron paramagnetic resonance (EPR). The phase transformations trends: hexagonal --> lamellar --> cubic --> hexagonal or hexagonal --> hexagonal, lamellar mixture --> cubic --> lamellar were observed by variations of the base or acid content of the gel or reaction temperature respectively. The results show at low surfactant /Si ratio, Mn-MCM-41 can be synthesized both in acid and base medium, and in a wide range of temperature (21-100 degrees C); Mn-MCM-48 can be obtained only in basic medium and in high temperature (100-120 degrees C); lamellar phase is formed in high temperature (>130 degrees C), and caused the collapse of the structure after calcination at 540 degrees C. Addition of Mn ions induces the formation of cubic phase also at low surfactant/Si ratio (0.12). By variations of the base content of the gel, Al-MCM-48 also can be synthesized. EPR results suggest that Mn ions can be incorporated into the amorphous silica wall of M41S or in the interface region of the polar head groups of the template. After calcination, Mn ions in Mn-MCM-41 and Mn-MCM-48, have high mobility and are not located within the inorganic wall.

The general expression for the echo intensity generated by the recently designed four-pulse electron-spin-echo envelope modulation experiment was derived, and a detailed analysis of this expression in terms of the relative contributions of the various terms to the electron-nuclear double-resonance frequencies and their combinations was performed. The terms contributing to the echo intensity were grouped according to the total number of generated EPR coherences in order to select those responsible for the combination harmonics. The latter are important since in orientationally disordered systems they exhibit better resolution than in the basic frequencies, and they can be used to determine the anisotropic hyperfine interaction and the nuclear quadrupole interaction. When the nuclear Zeeman interaction dominates the nuclear spin Hamiltonian, a significant number of terms in the final expression for the echo intensity have negligible contributions to the echo intensity, and they can be omitted to reduce computing time. A specific analysis was performed for the spin system S = 1/2, I = 5/2 in orientationally disordered samples with a large g anisotropy, which enables orientation-selective experiments. The lineshape and resolution of the combination peaks were explored through variations of the relative orientations of the g, hyperfine, and nuclear quadrupole tensors and the orientation of the external magnetic field. It is demonstrated that although the combination peaks are often dominated by the sum-combination harmonic corresponding to the nuclear \1/2]-\-1/2] transition, under several conditions of orientation selectivity it is possible to obtain combination lines split to five lines from which the quadrupolar interaction can be determined. (C) 1994 Academic Press, Inc.

The interactions of o-chloranyl (tetrachloro-1,2-benzoquinone) with Lewis acid sites in HY, H-mordenite and H-ZSM-5 zeolites and in aluminum oxide have been studied using electron spin resonance, electron nuclear double resonance and electron spin echo envelope modulation spectroscopies. The results are compared to those obtained from paramagnetic complexes generated in a solution of o-quinone and AlCl3 which serves as a model system. The detected Al-27 hyperfine interactions lead to the conclusion that adsorption of o-chloranyl on activated catalysts yields a paramagnetic complex with Al3+ Lewis acid sites. Hence, o-chloranyl can be used as an indicator for the detection and quantification of such sites.

Electron spin echo envelope modulation (ESEEM) induced by Al-27 nuclei can be used to characterize the interactions of paramagnetic transition metal cations with the framework of various aluminosilicates. The quantitative analysis of such systems is complex due to the Al-27 nuclear quadrupole interaction which is often large and unknown. In order to obtain a better understanding of the various spectral features of the Al-27 modulation, the ESEEM of a S = 1/2 I = 5/2 spin system in orientationally disordered systems was investigated in the frequency domain. The relative contributions of the various electron-nuclear double resonance (ENDOR) frequencies to the Fourier transform (FT) ESEEM spectrum were studied as a function of the size of the nuclear quadrupole coupling constant and of the relative orientation of the nuclear quadrupole tensor with respect to the g-tensor. The parameters range investigated is that expected from Cu2+ interacting with framework Al in zeolites. Two approaches were employed in the calculations, exact diagonalization of the nuclear Hamiltonian and a second order perturbation treatment of the quadrupole interaction. It is found that the second order perturbation approach applies up to e2qQ/h - \ - 1/2> ENDOR transitions were explored as well. This occurs when e2qQ/h is relatively large and it strongly depends on the orientation of the quadrupole tensor. When the FT-ESEEM can be described only by the \1/2> - \ - 1/2> ENDOR transitions, the simulations are significantly simplified since these can be calculated using the analytical expressions obtained by perturbation theory also for a quadrupole coupling constant as large as 10 MHz.

The interactions of Cu2+ cations with framework Al in zeolites NaX, KX, NaY, and NaA in various stages of dehydration and after methanol adsorption were investigated by electron spin echo envelope modulation (ESEEM) spectroscopy. The FT-ESEEM spectra of all species studies showed contributions from two types of Al-27 nuclei. The first type, referred to as first shell, consists of Al-27 nuclei bonded to the oxygens to which the Cu2+ is coordinated. They exhibit relatively large isotropic hyperfine constants which manifest the firm binding of the Cu2+ to the framework. The second type consists of Al nuclei which are coupled to the Cu2+ by weak dipolar interactions and are termed distant Al. Orientation selective ESEEM experiments were carried out as well to obtain additional structural information. The modulation amplitude corresponding to distant Al did not show any dependence on the irradiation position within the EPR powder pattern as the arrangement of those nuclei around the cation is approximately isotropic. The modulation amplitudes of the first shell Al in some cases showed strong orientation dependence which was interpreted in terms of the site geometry. The amount of adsorbed methanol in Cu-NaX has a considerable effect on the relative intensities of the peaks of the first shell Al and distant Al. This dependence is explained in terms of the changes in the Al-27 quadrupole coupling constants of the two types of Al.

The immediate environment of Mn(II) in MnAlPO5 after synthesis, calcination, hydration, and dehydration has been investigated by both conventional ESR spectroscopy and by several electron spin-echo (ESE) methods. Several samples with Mn(II) contents between Mn/P = 0.05 and 0.5 atom % were synthesized, and samples of impregnated Mn-AlPO5 and exchanged Mn-SAPO5 were used as references. The X-band and Q-band ESR spectra of all as-synthesized and calcined MnAlPO5 samples and of impregnated Mn-AlPO5 are characteristic of Mn(II) in a slightly distorted octahedral symmetry with an hyperfine coupling constant of 90 G and a zero-field splitting parameter of 140 G. The spatial distribution of the Mn(II) cations was investigated by the "2 + 1" ESE experiment. A random homogeneous distribution of isolated Mn(II) cations was found only in the sample with the lowest Mn(II) content. In all other samples regions with enriched Mn(II) concentrations, where most of the Mn(II) are located, were found to exist. Dehydration causes the migration of the Mn(II) cations toward each other, and consequently the spin exchange interaction increases, resulting in the averaging of the hyperfine interaction. Electron spin-echo envelope modulation (ESEEM) experiments, which detect only the isolated Mn(II) species, showed that the isolated Mn(II) cations interact through weak dipolar interactions with an average of 6 P-31 nuclei at a distance of 5 angstrom. This does not agree with a model of Mn(II) substituting for framework Al. Significant changes in the Al modulation after calcination and hydration indicated that this treatment results in a change in the Mn(II) location. Mn(II) in calcined and hydrated MnAlPO5 and in impregnated Mn-AlPO5 is coordinated to three or four framework oxygens, and the remaining ligands are water molecules and/or hydroxyl groups. All the above observations lead to the conclusion that the majority of the Mn(II) in MnAlPO5 does not occupy framework sites but is probably