Publications

Elucidating structural information of supramolecular hostguest systems is pivotal for understanding molecular recognition and designing functional materials. This study explores the binding modes of fluorinated benzylamine guests in cyclodextrin-based paramagnetic cavitands, employing Gd(III)-capped cyclodextrins (Gd-a-CD and Gd-b-CD, comprising six and seven glucopyranoside units, respectively) and high-field ¹⁹F electron-nuclear double resonance (ENDOR). The ¹⁹F ENDOR spectra revealed distinct behaviors based on the fluorine position and cyclodextrin cavity size. For para-fluorinated benzylamine guests, Gd-b-CD displayed a bimodal distribution of GdF distances, corresponding to two distinct binding modes, whereas Gd-a-CD exhibited a single binding mode. In contrast, meta-fluorinated benzylamines demonstrated a single binding mode for both Gd-a-CD and Gd-b-CD, underscoring the influence of cavity size and fluorine substitution in the guest on binding specificity. ENDOR measurements performed at the EPR central transition of Gd(III) are generally expected to yield GdF distances without orientation-specific details. Surprisingly, in Gd-CDs systems, an unexpected orientation selectivity was observed, enabling the extraction of both GdF distances and orientation of the guest molecule relative to the cavitands Gd(III) zero-field splitting (ZFS) tensor. This two-faceted capability of ¹⁹F-ENDOR allows for determining hostguest complexation geometry and provides insights into ZFS orientation within the cavitand structure.

¹⁹F electron-nuclear double resonance (ENDOR) has emerged as an attractive method for determining distance distributions in biomolecules in the range of 0.7-2 nm, which is not easily accessible by pulsed electron dipolar spectroscopy. The ¹⁹F ENDOR approach relies on spin labeling, and in this work, we compare various labels performance. Four protein variants of GB1 and ubiquitin bearing fluorinated residues were labeled at the same site with nitroxide and trityl radicals and a Gd(iii) chelate. Additionally, a double-histidine variant of GB1 was labeled with a Cu(ii) nitrilotriacetic acid chelate. ENDOR measurements were carried out at W-band (95 GHz) where ¹⁹F signals are well separated from ¹H signals. Differences in sensitivity were observed, with Gd(iii) chelates providing the highest signal-to-noise ratio. The new trityl label, OXMA, devoid of methyl groups, exhibited a sufficiently long phase memory time to provide an acceptable sensitivity. However, the longer tether of this label effectively reduces the maximum accessible distance between the ¹⁹F and the C_α of the spin-labeling site. The nitroxide and Cu(ii) labels provide valuable additional geometric insights via orientation selection. Prediction of electron-nuclear distances based on the known structures of the proteins were the closest to the experimental values for Gd(iii) labels, and distances obtained for Cu(ii) labeled GB1 are in good agreement with previously published NMR results. Overall, our results offer valuable guidance for selecting optimal spin labels for ¹⁹F ENDOR distance measurement in proteins.

Protein solutions can undergo liquid-liquid phase separation (LLPS), where a dispersed phase with a low protein concentration coexists with coacervates with a high protein concentration. We focus on the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein, CPEB4_NTD, and its isoform depleted of the Exon4, CPEB4Δ4_NTD. They both exhibit LLPS, but in contrast to most systems undergoing LLPS, the single-phase regime preceding LLPS consists mainly of soluble protein clusters. We combine experimental and theoretical approaches to resolve the internal structure of the clusters and the basis for their formation. Dynamic light scattering and atomic force microscopy show that both isoforms exhibit clusters with diameters ranging from 35 to 80 nm. Electron paramagnetic resonance spectroscopy of spin-labeled CPEB4_NTD and CPEB4Δ4_NTD revealed that these proteins have two distinct dynamical properties in both the clusters and coacervates. Based on the experimental results, we propose a core-shell structure for the clusters, which is supported by the agreement of the dynamic light scattering data on cluster size distribution with a statistical model developed to describe the structure of clusters. This model treats clusters as swollen micelles (microemulsions) where the core and the shell regions comprise different protein conformations, in agreement with the electron paramagnetic resonance detection of two protein populations. The effects of ionic strength and the addition of 1,6-hexanediol were used to probe the interactions responsible for cluster formation. While both CPEB4_NTD and CPEB4Δ4_NTD showed phase separation with increasing temperature and formed clusters, differences were found in the properties of the clusters and the coacervates. The data also suggested that the coacervates may consist of aggregates of clusters.

This chapter presents the application of electron paramagnetic resonance (EPR) spectroscopy to exploring the structure and dynamics of biomolecules, primarily proteins. Emphasis is given to pulsed dipolar EPR (PD-EPR), which provides distance distributions, typically in the range of 1.58 nm. Such measurements, usually carried out at low temperatures on frozen samples, are well established in solution and have been demonstrated as effective for in-cell studies. The chapter begins with a brief description of continuous wave EPR, followed by the most commonly used PD-EPR techniques, along with the associated data analysis methodology. Since most biomolecules are diamagnetic, site-directed spin labeling plays an essential role in EPR applications. The various spin labels currently available are discussed next. This is followed by a description of approaches used to model the rotational freedom of the spin label upon binding to specific sites in known structures and a prediction of the ensuing distance distribution. Then the use of distance distributions as constraints in structural derivation is briefly presented. The chapter concludes by showing some examples of the concurrent use of EPR techniques with other biophysical methods, such as NMR, SAXS, smFRET, and cryo-EM, highlighting actual biological applications, rather than model proteins often used for new method development.

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 paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with α-cysteine, penicillamine or β-cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electronelectron resonance experiments after titration with Gd3+ ions.

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

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 electronelectron 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.

MdfA from Escherichia coli is a prototypical secondary multi-drug (Mdr) transporter that exchanges drugs for protons. MdfA-mediated drug efflux is driven by the proton gradient and enabled by conformational changes that accompany the recruitment of drugs and their release. In this work, we applied distance measurements by W-band double electron-electron resonance (DEER) spectroscopy to explore the binding of mito-TEMPO, a nitroxide-labeled substrate analog, to Gd(III)-labeled MdfA. The choice of Gd(III)-nitroxide DEER enabled measurements in the presence of excess of mito-TEMPO, which has a relatively low affinity to MdfA. Distance measurements between mito-TEMPO and MdfA labeled at the periplasmic edges of either of three selected transmembrane helices (TM3¹⁰¹, TM5¹⁶⁸, and TM9³¹⁰) revealed rather similar distance distributions in detergent micelles (n-dodecyl-β-D-maltopyranoside, DDM)) and in lipid nanodiscs (ND). By grafting the predicted positions of the Gd(III) tag on the inward-facing (I_f) crystal structure, we looked for binding positions that reproduced the maxima of the distance distributions. The results show that the location of the mito-TEMPO nitroxide in DDM-solubilized or ND-reconstituted MdfA is similar (only 0.4 nm apart). In both cases, we located the nitroxide moiety near the ligand binding pocket in the I_f structure. However, according to the DEER-derived position, the substrate clashes with TM11, suggesting that for mito-TEMPO-bound MdfA, TM11 should move relative to the I_f structure. Additional DEER studies with MdfA labeled with Gd(III) at two sites revealed that TM9 also dislocates upon substrate binding. Together with our previous reports, this study demonstrates the utility of Gd(III)-Gd(III) and Gd(III)-nitroxide DEER measurements for studying the conformational behavior of transporters.

Gd(III) complexes are currently established as spin labels for structural studies of biomolecules using pulse dipolar electron paramagnetic resonance (PD-EPR) techniques. This has been achieved by the availability of medium- and high-field spectrometers, understanding the spin physics underlying the spectroscopic properties of high spin Gd(III) (S = 7/2) pairs and their dipolar interaction, the design of well-defined model compounds and optimization of measurement techniques. In addition, a variety of Gd(III) chelates and labeling schemes have allowed a broad scope of applications. In this review, we provide a brief background of the spectroscopic properties of Gd(III) pertinent for effective PD-EPR measurements and focus on the various labels available to date. We report on their use in PD-EPR applications and highlight their pros and cons for particular applications. We also devote a section to recent in-cell structural studies of proteins using Gd(III), which is an exciting new direction for Gd(III) spin labeling.

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.

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the Antennapedia homeodomain orthogonally labeled with Gcl(3+) and Mn2+ tags in complex with its cognate DNA binding site labeled with a nitroxide.

Chirp and shaped pulses have been recently shown to be highly advantageous for improving sensitivity in DEER (double electronelectron resonance, also called PELDOR) measurements due to their large excitation bandwidth. The implementation of such pulses for pulse EPR has become feasible due to the availability of arbitrary waveform generators (AWG) with high sampling rates to support pulse shaping for pulses with tens of nanoseconds duration. Here we present a setup for obtaining chirp pulses on our home-built W-band (95 GHz) spectrometer and demonstrate its performance on Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements. We carried out an extensive optimization procedure on two model systems, Gd(III)-PyMTAspacerGd(III)-PyMTA (Gd-PyMTA ruler; zero-field splitting parameter (ZFS) D ∼ 1150 MHz) as well as nitroxidespacernitroxide (nitroxide ruler) to evaluate the applicability of shaped pulses to Gd(III) complexes and nitroxides, which are two important classes of spin labels used in modern DEER/EPR experiments. We applied our findings to ubiquitin, doubly labeled with Gd-DOTA-monoamide (D ∼ 550 MHz) as a model for a system with a small ZFS. Our experiments were focused on the questions (i) what are the best conditions for positioning of the detection frequency, (ii) which pump pulse parameters (bandwidth, positioning in the spectrum, length) yield the best signal-to-noise ratio (SNR) improvements when compared to classical DEER, and (iii) how do the sample's spectral parameters influence the experiment. For the nitroxide ruler, we report an improvement of up to 1.9 in total SNR, while for the Gd-PyMTA ruler the improvement was 3.13.4 and for Gd-DOTA-monoamide labeled ubiquitin it was a factor of 1.8. Whereas for the Gd-PyMTA ruler the two setups pump on maximum and observe on maximum gave about the same improvement, for Gd-DOTA-monoamide a significant difference was found. In general the choice of the best set of parameters depends on the D parameter of the Gd(III) complex.

We have applied high-field (W-band) pulse electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (EDNMR) to characterize the coordination sphere of the Mn²⁺ co-factor in the nucleotide binding sites (NBSs) of ABC transporters. MsbA and BmrCD are two efflux transporters hypothesized to represent divergent catalytic mechanisms. Our results reveal distinct coordination of Mn²⁺ to ATP and transporter residues in the consensus and degenerate NBSs of BmrCD. In contrast, the coordination of Mn²⁺ at the two NBSs of MsbA is similar, which provides a mechanistic rationale for its higher rate constant of ATP hydrolysis relative to BmrCD. Direct detection of vanadate ion, trapped in a high-energy post-hydrolysis intermediate, further supports the notion of asymmetric hydrolysis by the two NBSs of BmrCD. The integrated spectroscopic approach presented here, which link energy input to conformational dynamics, can be applied to a variety of systems powered by ATP turnover.

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper-sulfur (Cu-S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared³³S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron-electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the³³S nuclear frequencies, from which the hyperfine coupling and the spin population on the sulfur of the thiolate ligand were derived. The overlapping³³S and¹⁴N EDNMR signals were resolved using a recently introduced two-dimensional correlation technique, 2D-EDNMR. The³³S hyperfine tensor was determined by simulations of the EDNMR spectra using³³S hyperfine and quadrupolar tensors predicted by QM/MM DFT calculations as starting points for a manual spectral fit procedure. To reach a reasonable agreement with the experimental spectra, the³³S hyperfine principal value, A_z, and one of the corresponding Euler angles had to be modified. The final values obtained gave an experimentally determined sulfur spin population of 29.8 ± 0.7%,significantly improving the wide range of 29-62% reported in the literature. Our direct, experimentally derived value now provides an important constraint for further theoretical work aimed at unravelling the unique electronic properties of this site.

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.

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 (μRFQ) 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 3,5-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ 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 r AO + L r BO r 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 μRFQ 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.

Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-β component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δ_χ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd³⁺-Gd³⁺ distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δ_χ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm³⁺ and C3-Tb³⁺ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb³⁺ and Tm³⁺ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.

Although Gd³⁺-based spin labels have been shown to be an alternative to nitroxides for double electron-electron resonance (DEER) distance measurements at high fields, their ability to provide solvent accessibility information, as nitroxides do, has not been explored. In addition, the effect of the label type on the measured distance distribution has not been sufficiently characterized. In this work, we extended the applicability of Gd³⁺ spin labels to solvent accessibility measurements on a peptide in model membranes, namely, large unilamellar vesicles (LUVs) using W-band ²H Mims electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) techniques and Gd³⁺-ADO3A-labeled melittin. In addition, we carried out Gd³⁺-Gd³⁺ DEER distance measurements to probe the peptide conformation in solution and when bound to LUVs. A comparison with earlier results reported for the same system with nitroxide labels shows that, although in both cases the peptide binds parallel to the membrane surface, the Gd³⁺-ADO3A label tends to protrude from the membrane into the solvent, whereas the nitroxide does the opposite. This can be explained on the basis of the hydrophilicity of the Gd³⁺-ADO3A labels in contrast with the hydrophobicity of nitroxides. The distance distributions obtained from different labels are accordingly different, with the Gd³⁺-ADO3A yielding consistently broader distributions. These discrepancies are most pronounced when the peptide termini are labeled, which implies that such labeling positions may be inadvisible.

Gd(iii) complexes have emerged as spin labels for distance determination in biomolecules through double-electron-electron resonance (DEER) measurements at high fields. For data analysis, the standard approach developed for a pair of weakly coupled spins with S = 1/2 was applied, ignoring the actual properties of Gd(iii) ions, i.e. S = 7/2 and ZFS (zero field splitting) ≠ 0. The present study reports on a careful investigation on the consequences of this approach, together with the range of distances accessible by DEER with Gd(iii) complexes as spin labels. The experiments were performed on a series of specifically designed and synthesized Gd-rulers (Gd-PyMTA-spacer-Gd-PyMTA) covering Gd-Gd distances of 2-8 nm. These were dissolved in D₂O-glycerol-d₈ (0.03-0.10 mM solutions) which is the solvent used for the corresponding experiments on biomolecules. Q- and W-band DEER measurements, followed by data analysis using the standard data analysis approach, used for S = 1/2 pairs gave the distance-distribution curves, of which the absolute maxima agreed very well with the expected distances. However, in the case of the short distances of 2.1 and 2.9 nm, the distance distributions revealed additional peaks. These are a consequence of neglecting the pseudo-secular term in the dipolar Hamiltonian during the data analysis, as is outlined in a theoretical treatment. At distances of 3.4 nm and above, disregarding the pseudo-secular term leads to a broadening of a maximum of 0.4 nm of the distance-distribution curves at half height. Overall, the distances of up to 8.3 nm were determined, and the long evolution time of 16 μs at 10 K indicates that a distance of up to 9.4 nm can be accessed. A large distribution of the ZFS parameter, D, as is found for most Gd(iii) complexes in a frozen solution, is crucial for the application of Gd(iii) complexes as spin labels for distance determination via Gd(iii)-Gd(iii) DEER, especially for short distances. The larger ZFS of Gd-PyMTA, in comparison to that of Gd-DOTA, makes Gd-PyMTA a better label for short distances.

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.

In dynamic nuclear polarisation (DNP) experiments performed under static conditions at 1.4 K we show that the presence of 1 mM Gd(iii)-DOTAREM increases the ¹³C polarisation and decreases the ¹³C polarisation buildup time of ¹³C-urea dissolved in samples containing water/DMSO mixtures with trityl radical (OX063) concentrations of 10 mM or higher. To account for these observations further measurements were carried out at 6.5 K, using a combined EPR and NMR spectrometer. At this temperature, frequency swept DNP spectra of samples with 5 or 10 mM OX063 were measured, with and without 1 mM Gd-DOTA, and again a ¹³C enhancement gain was observed due to the presence of Gd-DOTA. These measurements were complemented by electron-electron double resonance (ELDOR) measurements to quantitate the effect of electron spectral diffusion (eSD) on the DNP enhancements and lineshapes. Simulations of the ELDOR spectra were done using the following parameters: (i) a parameter defining the rate of the eSD process, (ii) an "effective electron-proton anisotropic hyperfine interaction parameter", and (iii) the transverse electron spin relaxation time of OX063. These parameters, together with the longitudinal electron spin relaxation time, measured by EPR, were used to calculate the frequency profile of electron polarisation. This, in turn, was used to calculate two basic solid effect (SE) and indirect cross effect (iCE) DNP spectra. A properly weighted combination of these two normalized DNP spectra provided a very good fit of the experimental DNP spectra. The best fit simulation parameters reveal that the addition of Gd(iii)-DOTA causes an increase in both the SE and the iCE contributions by similar amounts, and that the increase in the overall DNP enhancements is a result of narrowing of the ELDOR spectra (increased electron polarisation gradient across the EPR line). These changes in the electron depolarisation profile are a combined result of shortening of the longitudinal and transverse electron spin relaxation times, as well as an increase in the eSD rate and in the effective electron-proton anisotropic hyperfine interaction parameter.

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 multispin 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.

The microwave frequency swept DNP enhancement, referred to as the DNP spectrum, is strongly dependent on the EPR spectrum of the polarizing radical and it reveals the underlying DNP mechanisms. Here we focus on two chlorinated trityl radicals that feature axially symmetric powder patterns at 95 GHz, the width of which are narrower than those of TEMPOL or TOTAPOL but broader than that of the trityl derivative OX63. The static DNP lineshapes of these commonly used radicals in DNP, have been recently analyzed in terms of a superposition of basic Solid Effect (SE) and Cross Effect (CE)-DNP lineshapes, with their relative contributions as a fit parameter. To substantiate the generality of this approach and further investigate an earlier suggestion that a ^35,37Cl-¹³C polarization transfer pathway, termed \u201chetero-nuclear assisted DNP\u201d, may be in effect in the chlorinated radicals (C. Gabellieri et al., Angew. Chem., Int. Ed., 2010, 49, 3360-3362), we measured the static ¹³C-glycerol DNP spectra of solutions of ca.∼10 mM of the two chlorinated trityl radicals as a function of temperature (10-50 K) and microwave power. Analysis of the DNP lineshapes was first done in terms of the SE/CE superposition model calculated assuming a direct e-¹³C polarization transfer. The CE was found to prevail at the high temperature range (40-50 K), whereas at the low temperature end (10-20 K) the SE dominates, as was observed earlier for ¹³C DNP with OX63 and ¹H DNP with TEMPOL and TOTAPOL, thus indicating that this is rather general behavior. Furthermore, it was found that at low temperatures it is possible to suppress the SE, and increase the CE by merely lowering the microwave power. While this analysis gave a good agreement between experimental and calculated lineshapes when the CE dominates, some significant discrepancies were observed at low temperatures, where the SE dominates. We show that by explicitly taking into account the presence of ^35/37Cl nuclei through a e-^35,37Cl-¹³C polarization pathway in the SE-DNP lineshape calculations, as proposed earlier, we can improve the fit significantly, thus supporting the existence of the \u201chetero-nuclear assisted DNP\u201d pathway.

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 ∼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.

ELDOR (Electron Double Resonance)-detected NMR (EDNMR) is a pulse EPR experiment that is used to measure the transition frequencies of nuclear spins coupled to electron spins. These frequencies are further used to determine hyperfine and quadrupolar couplings, which are signatures of the electronic and spatial structures of paramagnetic centers. In recent years, EDNMR has been shown to be particularly useful at high fields/high frequencies, such as W-band (∼95 GHz, ∼3.5 T), for low γ quadrupolar nuclei. Although at high fields the nuclear Larmor frequencies are usually well resolved, the limited resolution of EDNMR still remains a major concern. In this work we introduce a two dimensional, triple resonance, correlation experiment based on the EDNMR pulse sequence, which we term 2D-EDNMR. This experiment allows circumventing the resolution limitation by spreading the signals in two dimensions and the observed correlations help in the assignment of the signals. First we demonstrate the utility of the 2D-EDNMR experiment on a nitroxide spin label, where we observe correlations between ¹⁴N nuclear frequencies. Negative cross-peaks appear between lines belonging to different M_S electron spin manifolds. We resolved two independent correlation patterns for nuclear frequencies arising from the EPR transitions corresponding to the ¹⁴N m_I = 0 and m_I = -1 nuclear spin states, which severely overlap in the one dimensional EDNMR spectrum. The observed correlations could be accounted for by considering changes in the populations of energy levels that S = 1/2, I = 1 spin systems undergo during the pulse sequence. In addition to these negative cross-peaks, positive cross-peaks appear as well. We present a theoretical model based on the Liouville equation and use it to calculate the time evolution of populations of the various energy levels during the 2D-EDNMR experiment and generated simulated 2D-EDMR spectra. These calculations show that the positive cross-peaks appear due to off resonance effects and/or nuclear relaxation effects. These results suggest that the 2D-EDNMR experiment can be also useful for relaxation pathway studies. Finally we present preliminary results demonstrating that 2D-EDNMR can resolve overlapping ³³S and ¹⁴N signals of type 1 Cu(II) center in ³³S enriched Azurin.

Nitroxide spin-labelled lipid analogues are often used to study model membrane properties using EPR spectroscopy. Whereas in liquid phase membranes the spin label assumes, on average, its putative location, in gel phases and frozen membrane, depending on its position along the acyl chain, it may exhibit a different average location. Here we used ²H three-pulse Electron Spin Echo Envelope Modulation (ESEEM) of phospholipid spin probes, combined with various deuteration schemes to detect the effect of the model membrane curvature and cholesterol on vertical migrations of the spin label. We compared large and small unilamellar 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with and without cholesterol (10%). The vertical displacement of the spin label was manifested as an apparently flat trans-membrane profile of water concentration and of label proximity to the head group choline. The spin-label propensity to migrate was found to increase with vesicle curvature and decrease in the presence of cholesterol. This in turn reflects the effect of packing and ordering of the membrane lipids. The results show that in curved vesicles lacking cholesterol, the label attached to carbon 16 may travel as far high along the membrane normal as the location of the label on carbon 5, due to the presence of U-shaped lipid conformations. This phenomenon must be taken into account when using spin-labelled lipids as membrane depth markers or to trace trans-membrane profiles.

Interspin distances between 0.8 nm and 2.0 nm can be measured through the dipolar broadening of the continuous wave (cw) EPR spectrum of nitroxide spin labels at X-band (9.4 GHz, 0.35 T). We introduce Gd³⁺ as a promising alternative spin label for distance measurements by cw EPR above 7 Tesla, where the 1/2〉 to 1/2〉 transition narrows below 1 mT and becomes extremely sensitive to dipolar broadening. To estimate the distance limits of cw EPR with Gd³⁺, we have measured spectra of frozen solutions of GdCl ₃ at 8.6 T (240 GHz) and 10 K at concentrations ranging from 50 mM to 0.1 mM, covering a range of average interspin distances. These experiments show substantial dipolar broadening at distances where line broadening cannot be observed with nitroxides at X-band. This data, and its agreement with calculated dipolar-broadened lineshapes, show Gd³⁺ to be sensitive to distances as long as ∼3.8 nm. Further, the linewidth of a bis-Gd³⁺ complex with a flexible ∼1.6 nm bridge is strongly broadened as compared to the mono-Gd³⁺ complex, demonstrating the potential for application to pairwise distances. Gd-DOTA-based chelates that can be functionalized to protein surfaces display linewidths narrower than aqueous GdCl₃, implying they should be even more sensitive to dipolar broadening. Therefore, we suggest that the combination of tailored Gd³⁺ labels and high magnetic fields can extend the longest interspin distances measurable by cw EPR from 2.0 nm to 3.8 nm. cw EPR data at 260 K demonstrate that the line broadening remains clear out to similar average interspin distances, offering Gd³⁺ probes as promising distance rulers at temperatures higher than possible with conventional pulsed EPR distance measurements.

Rapid freeze quench electron paramagnetic resonance (RFQ)-EPR is a method for trapping short lived intermediates in chemical reactions and subjecting them to EPR spectroscopy investigation for their characterization. Two (or more) reacting components are mixed at room temperature and after some delay the mixture is sprayed into a cold trap and transferred into the EPR tube. A major caveat in using commercial RFQ-EPR for high field EPR applications is the relatively large amount of sample needed for each time point, a major part of which is wasted as the dead volume of the instrument. The small sample volume (∼2 μl) needed for high field EPR spectrometers, such as W-band (∼3.5 T, 95 GHz), that use cavities calls for the development of a microfluidic based RFQ-EPR apparatus. This is particularly important for biological applications because of the difficulties often encountered in producing large amounts of intrinsically paramagnetic proteins and spin labeled nucleic acid and proteins. Here we describe a dedicated microfluidic based RFQ-EPR apparatus suitable for small volume samples in the range of a few μl. The device is based on a previously published microfluidic mixer and features a new ejection mechanism and a novel cold trap that allows collection of a series of different time points in one continuous experiment. The reduction of a nitroxide radical with dithionite, employing the signal of Mn²⁺ as an internal standard was used to demonstrate the performance of the microfluidic RFQ apparatus.

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 α-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 ²H 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 ²H nuclei in the hydrophilic region of the membrane and the density of the ²H 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 ²H 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.

Hard-ligand, high-potential copper sites have been characterized in double mutants of Pseudomonas aeruginosa azurin (C112D/M121X (X = L, F, I)). These sites feature a small A _zz(Cu) splitting in the EPR spectrum together with enhanced electron transfer activity. Due to these unique properties, these constructs have been called "type zero" copper sites. In contrast, the single mutant, C112D, features a large A _zz(Cu) value characteristic of the typical type 2 Cu ^II. In general, A _zz(Cu) comprises contributions from Fermi contact, spin dipolar, and orbital dipolar terms. In order to understand the origin of the low A _zz(Cu) value of type zero Cu ^II, we explored in detail its degree of covalency, as manifested by spin delocalization over its ligands, which affects A _zz(Cu) through the Fermi contact and spin dipolar contributions. This was achieved by the application of several complementary EPR hyperfine spectroscopic techniques at X- and W-band (∼9.5 and 95 GHz, respectively) frequencies to map the ligand hyperfine couplings. Our results show that spin delocalization over the ligands in type zero Cu ^II is different from that of type 2 Cu ^II in the single C112D mutant. The ¹⁴N hyperfine couplings of the coordinated histidine nitrogens are smaller by about 25-40%, whereas that of the ¹³C carboxylate of D112 is about 50% larger. From this comparison, we concluded that the spin delocalization of type zero copper over its ligands is not dramatically larger than in type 2 C112D. Therefore, the reduced A _zz(Cu) value of type zero Cu ^II is largely attributable to an increased orbital dipolar contribution that is related to its larger g _zz value, as a consequence of the distorted tetrahedral geometry. The increased spin delocalization over the D112 carboxylate in type zero mutants compared to type 2 C112D suggests that electron transfer paths involving this residue are enhanced.

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.

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 polar head and alkyl chain regions with increasing P/PL; while the DPPC density increased in the polar head region, it decreased in the alkyl chain region. These results are consistent with disruption of the lipid order and segregation of the PL constituents of the membrane as a consequence of the melittin binding. This work further demonstrates the applicability and potential of pulse EPR techniques for the study of peptide-membrane interactions.

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the proteins ATPase site in solution. Specifically, we substituted the essential Mg ²⁺ cofactor in the ATPase active site for paramagnetic Mn ²⁺ and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn ²⁺ interactions with the nucleotide phosphates through the ³¹P hyperfine couplings and the coordination by protein residues through ¹³C hyperfine coupling from ¹³C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the ¹³C and the ³¹P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn ²⁺ ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn ²⁺ cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the proteins ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn ²⁺ ion. Finally, a competitive Mn ²⁺ binding site was found for single-stranded RNA construct.

Double electron-electron resonance (DEER) distance measurements of a protein complex tagged with two Gd³⁺ chelates developed for rigid positioning of the metal ion are shown to deliver outstandingly accurate distance measurements in the 6 nm range. The accuracy was assessed by comparison with modeled distance distributions based on the three-dimensional molecular structures of the protein and the tag and further comparison with paramagnetic NMR data. The close agreement between the predicted and experimentally measured distances opens new possibilities for investigating the structure of biomolecular assemblies. As an example, we show that the dimer interface of rat ERp29 in solution is the same as that determined previously for human ERp29 in the single crystal.

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 ¹⁴N 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 ¹⁴N 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 ¹⁴N 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 ¹⁴N 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 |P_zz| decreases with polarity, as predicted by Savitsky et al.

cd₁ nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d₁-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 d₁-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 d₁-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d₁-heme NO complex from Pseudomonas aeruginosa cd₁ 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 (Tyr₁₀, His₃₂₇, and His₃₆₉), 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 (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d₁-heme complex of cd₁ NIR forms H-bonds with Tyr₁₀ and His₃₆₉, whereas the second conserved histidine, His₃₂₇, appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr₁₀ 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 shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His₃₆₉ is closer to the NO, whereas mutation of both distal histidines displaces Tyr ₁₀, removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis 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 , allowing us to probe the shortest limits of accessible distances which were found to be as small as 13 . The upper distance limit for these labels was also evaluated and was found to be about 60 . 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.

Self-assembly (SA) of amphophilic block copolymers (polyethylene 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. Nitroxidelabeled 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 (with a 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 present a new approach to obtain details on the distribution and average structure and locations of membrane-associated peptides. The approach combines (i) pulse double electron-electron resonance (DEER) to determine intramolecular distances between residues in spin labeled peptides, (ii) electron spin echo envelope modulation (ESEEM) experiments to measure water exposure and the direct interaction of spin labeled peptides with deuterium nuclei on the phospholipid molecules, and (iii) Monte Carlo (MC) simulations to derive the peptide-membrane populations, energetics, and average conformation of the native peptide and mutants mimicking the spin labeling. To demonstrate the approach, we investigated the membrane-bound and solution state of the well-known antimicrobial peptide melittin, used as a model system. A good agreement was obtained between the experimental results and the MC simulations regarding the distribution of distances between the labeled amino acids, the side chain mobility, and the peptide's orientation. A good agreement in the extent of membrane penetration of amino acids in the peptide core was obtained as well, but the EPR data reported a somewhat deeper membrane penetration of the termini compared to the simulations. Overall, melittin adsorbed on the membrane surface, in a monomelic state, as an amphipatic helix with its hydrophobic residues in the hydrocarbon region of the membrane and its charged and polar residues in the lipid headgroup region.

The H₅PV₂Mo₁₀O₄₀ polyoxometalate and Pd/AI₂O₃ were used as co-catalysts under anaerobic conditions for the activation and oxidation of CO to CO ₂ by an electron transfer-oxygen transfer mechanism. Upon anaerobic reduction of H₅PV₂Mo₁₀O₄₀ with CO in the presence of Pd(0) two paramagnetic species were observed and characterized by continuous wave electron paramagnetic resonance (CW-EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopic measurements. Major species I (65-70%) is assigned to a species resembling a vanadyl cation that is supported on the polyoxometalate and showed a bonding interaction with ¹³CO. Minor species II (30-35%) is attributed to a reduced species where the vanadium(IV) atom is incorporated in the polyoxometalate framework but slightly distanced from the phosphate core. Under aerobic conditions, CO/O₂, a nucleophilic oxidant was formed as elucidated by oxidation of thianthrene oxide as a probe substrate. Oxidation reactions performed on terminal alkenes such as 1-octene yielded a complicated mixture of products that was, however, clearly a result of alkene epoxidation followed by subsequent reactions of the intermediate epoxide. The significant competing reaction was a hydro-carbonylation reaction that yielded a ∼1:1 mixture of linear/branched carboxylic acids.

Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate intramolecular and intermolecular complexes of cyclodextrins (CDs) with a nitroxide group. The interaction with solvent molecules (D₂O) was followed the ²H modulation depth. Competition experiments with adamantane-type guests were used to confirm complexation. The shielding of the nitroxide group from solvent upon inclusion into a CD cavity made this technique more sensitive to complexation thcw EPR spectroscopy. ESEEM analysis of a series of CDs mono and bis spin-labeled on the primary rim of the cavity showed that only one compound formed a self-inclusion complex. This suggests that significant linker length/flexibility is required for forof inclusion complexes in functionalized CDs. DEER (double electron-electron resonance) experiments confirmed that the self-inclusion complex of the spin-labeled CD was intra- rather than intermolecular.

The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates π pulses of 25 ns and a microwave field, B₁ = 0.71 mT. Such a high B₁ enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of ¹⁴N and ¹⁷O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides ¹⁴N quadrupolar splittings.

The self-assembly (SA) of amphiphilic block copolymers (poly(ethylene oxide)-poly(propylene oxide)-polyethylene 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 ¹⁴N isotropic hyperfine coupling, a_iso, and the rotational correlation time, τ_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-polymer 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)₂L^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)₂L^R breathing crystals interesting and promising systems in the research toward creation of molecular-magnetic switches and related spin devices.

A new, triple resonance, pulse electron paramagnetic resonance (EPR) sequence is described. It provides spin links between forbidden electron spin transitions (Δ MS =±1, Δ MI 0) and allowed nuclear spin transitions (Δ MI =±1), thus, facilitating the assignment of nuclear frequencies to their respective electron spin manifolds and paramagnetic centers. It also yields the relative signs of the hyperfine couplings of the different nuclei. The technique is based on the combination of electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected NMR experiments in a way similar to the TRIPLE experiment. The feasibility and the information content of the method are demonstrated first on a single crystal of Cu-doped L-histidine and then on a frozen solution of a Cu-histidine complex.

The addition of NaNO₃ to the reaction mixture of hexagonal SBA-15 changes the product to a Ia3̄d cubic phase. To understand the specific action of the salt, electron-spin echo envelope modulation (ESEEM) spectroscopy was applied. It allowed us to follow the local concentration of the individual ions within the micelles through their dipolar interaction with spin probes introduced into different regions of the Pluronic P123 (PEO ₂₀PPO₇₀PEO₂₀) micelles. The ¹⁵N and ²³Na modulation depth probed the local concentration profile of the anions and cations, and the water content in these regions was followed through the modulation depth of ²H in D₂O solutions. The ions were found to penetrate the hydrophilic corona region (the polyethylene oxide, PEO) of the micelles, concomitant with penetration of water molecules, reaching saturation at a bulk NaNO₃ concentration of 0.2 M. In acidic micellar solutions, protons repel the Na⁺ ions from the corona and for the same total [NO₃^-] the acidity increases the NO ₃^- capacity of the corona. However, a general decrease is noted in the nitrate and the water content of the corona with an increasing salt concentration in the bulk solution. This effective dehydration of the EO groups decreases the curvature of the micellar assembly of the Pluronic, and leads to the formation of the final cubic phase rather than the hexagonal phase. Time-resolved freeze quench ESEEM measurements, carried out on the reaction mixtures of the hexagonal and cubic phases, show that in both cases the nitrate concentration within the corona is reduced with time due to depletion of the protons through exchange with positively charged silica precursors. These results show that ESEEM measurements on Pluronic-like spin probes offer a new molecular level, quantitative method to observe the variations in location and amounts of the cations and anions within micelles.

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_∥) to high (g_⊥) 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_⊥ region, since the number of distant electrons able to participate in this relaxation mechanism is higher than in the g _∥ 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.

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 ³¹P ENDOR (electron-nuclear double resonance) measurements indicated the absence of a phosphorous heteroatom leading to the suggestion that H₅V^VV^IVMo₁₁O₄₀ exists as a previously unrecognized impurity in the typically synthesized H₅PV₂Mo₁₀O₄₀ compound. H_5/4PV^VO₄V^IV/VMo₁₁O₃₆ 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 [V^VV^IVMo₁₁O₄₀]^5- as [V^VO₄V^IVMo₁₁O₃₆]^5-. Accordingly, the polyoxometalate has a multiscripts(VO, 4, mml:none(), mml:none(), 3 -) 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 determination of the details of the spatial and electronic structure of functional sites (centers) in any system, be it in materials chemistry or in biology, is the first step towards understanding their function. When such sites happen to be paramagnetic in any point of their activity cycle, the tool box offered by a variety of high resolution electron paramagnetic resonance (EPR) spectroscopic techniques becomes very attractive for their characterization. This tool box has been considerably expanded by the developments in high field (HF) EPR in general, and HF electron nuclear double resonance (ENDOR), in particular. These have led to numerous new applications in the fields of biology, physics, chemistry and materials sciences. This overview focuses specifically on recent applications of pulsed HF ENDOR spectroscopy to microporous materials, such as zeotype materials, presenting the new opportunities it offers. First, a brief description of the theoretical basis required for the analysis of the HF ENDOR spectrum is given, followed by a description of the pulsed techniques used to record spectra and assign the signals, along with a brief presentation of the required instrumentation. Next, specific applications are given, including transition metal ions and complexes exchanged into zeolite cages, transition metal substitution into frameworks of zeolites, aluminophosphate molecular sieves, and silicious mesoporous materials, the interaction of NO with Lewis sites in zeolite cages and trapped S ^-₃ . We end with a discussion of the advantages and the shortcomings of the method and conclude with a future outlook.

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 °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 °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 profile 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 (7:3 w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3 w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the ²H 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_A²⁺ and Mn_B²⁺, 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. ¹H 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_A²⁺ and Mn _B²⁺. ⁵⁵Mn ENDOR spectra, which are dominated by the ⁵⁵Mn 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.

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.

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 a position and in a reaction carried out in D₂O. Comparing the deuterium modulation depth, k(²H), induced by CTAB-α-d₂, CTAB-d₉, or D₂O 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 mesostructure. Similar experiments carried out on reaction mixtures containing SL1SiEt showed a decrease of k(²H) from CTAB-α-d₂ and CTAB-d₉ compared to the micellar solution, exhibiting practically no time dependence. This indicates that the spin probe is pulled away from the micelle-water interface into the loosely linked, forming silica network. After drying, the modulation depth induced by CTAB-α-d₂ and CTAB-d₉ increases, showing that, once the water is removed, the silica walls contract around the micelles, pushing the silica-linked spin probe into the organic phase within the mesopores.

In this work we have studied pulsed ¹⁷O electron nuclear double resonance (ENDOR) spectra of the Gd ³⁺ aquo ion and the magnetic resonance imaging (MRI) contrast agent MS-325 in an ¹⁷O-enriched frozen glassy water/methanol solution. The isotropic hyperfine interaction (hfi) constant of the water ligand ¹⁷O was found to be about 0.75 MHz, which corresponds to a spin density delocalized to the ligand of ρ _o ≈ -4 × 10 ^-3. The analysis of the anisotropic hfi constant (0.69 ± 0.05 MHz) yields Gd-O distances of about 2.4-2.5 Å. Simultaneous analysis of these distances and the Gd-H distances found earlier allows one to elucidate the details of the Gd-OH ₂ coordination geometry.

A new and easy method for preparing blue sodalite pigments which involves high-temperature calcination of sodalite samples synthesized with aluminum sulfate and an organic template, is presented. Calcination generated the S ₃^-and S₂^-radicals, and the effects of the Al/Si ratio and the calcination temperature on the nature and amounts of the radicals were examined. The radicals were characterized in detail by continuous wave and pulsed EPR at X- and W-band frequencies (∼9 and 95 GHz, respectively) complemented by UV-vis measurements. The high-field electron-paramagnetic resonance (EPR) measurements allowed us to clearly resolve the g anisotropy of S₃^-and W-band electron nuclear double resonance (ENDOR) measurements detected strong coupling with extra-framework ²³Na cations and weak coupling with framework ²⁷AI. On the basis of the spectroscopic results and density functional theory (DFT) calculations of the g-tensors of S₃^-and S₂^-radicals, the EPR signals were attributed to three different S₃^-radicals, all with the open structure C_2v, that are located within the sodalite β ages. While two of these radicals are well isolated, the third one is associated with an exchange-narrowed signal originating from S₃^-radicals in nearby sodalite cages.

The structural features of Mn(II) incorporated into two large cage zeotypes, Mn-UCSB-10Mg and Mn-UCSB-6Mg, were explored by combining multifrequency CW-EPR with W-band ENDOR spectroscopy. As-synthesized samples, dried both at room temperature and 150°C, were examined and the results were compared with the reference samples Mn-AlPO₄-20 and Mn-AlPO ₄-5. High-frequency CW EPR experiments (at W- and G- bands) resolved two main types of Mn(II) framework sites with significantly different ⁵⁵Mn hyperfine couplings and slightly different g values. These results were further corroborated by ⁵⁵Mn ENDOR spectra, which enabled a more accurate determination of the two hyperfine coupling values and revealed the presence of a third species. ENDOR experiments carried out at magnetic fields away from the central, |-1/2,m_I〉 → |+1/2,m_I〉 EPR transitions, established a negative sign for A_iso(⁵⁵Mn). By comparison with as-synthesized samples that were mildly dehydrated the various species were assigned to framework sites with different degrees of water coordination. While one species is similar to the distorted (pseudo) tetrahedral sites found in the reference Mn-AlPO₄-20,5 samples, the other two experience interaction with weakly bound water ligands. The transformation between the three types upon dehydration and rehydration is reversible. In an attempt to improve the spectral resolution, W-band EPR and ENDOR measurements were carried out on single-crystals of Mn-UCSB-10Mg (typical size of ∼0.02 mm³). Similar to the polycrystalline sample, two main A_iso(⁵⁵Mn) components were resolved in the EPR spectra, their relative populations, however, differed from that of the polycrystalline material. This difference is attributed to variations in the water content originating from a crystal size effect. Surprisingly, the single crystal spectra did not show better resolution, and moreover, they did not exhibit significant orientation dependence in most of the experiments. These findings are ascribed to the presence of several chemically distinguishable sites combined with the multiplicity of symmetry related tetrahedral sites in one zeotype unit cell. Such a situation leads to an effective 'powder like' spectrum due to the small anisotropy of the magnetic interactions involved.

Two current frontiers in EPR research are high-field (ν₀ > 70 GHz, B₀ > 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 (∼9.5 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.

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 10⁷-10⁸ 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.

The incorporation of low levels of Mn(II) (Mn/Al∼0.001) into five aluminophosphate zeotypes was studied by high-field echo-detected EPR, and by ³¹p and ¹H electron-nuclear double resonance (ENDOR) spectroscopies. The zeotype structures investigated-SOD, AEL, AFI, SBS, and SBT-cover a variety of channel morphologies, and span a range of framework densities. The highly resolved EPR spectra could distinguish between two types of Mn with different ⁵⁵Mn hyperfine couplings in structures containing more than one T site. Mims and Davies ³¹p ENDOR spectra, recorded at a field set to one of the |-1/2, m_I| + 1/2, m_I) ⁵⁵Mn hyperfine components consist of a symmetric doublet, with a splitting in the range of 5-8 MHz. The large open structures showed smaller couplings than the denser morphologies. A similar ³¹p hyperfine was also detected for Fe(III) incorporated into aluminophosphate zeotype with the SOD structure. Variations in the IH ENDOR spectra of the various Mn(II) substituted zeotypes, particularly in the relative intensity of the ¹H matrix line, were detected as well. These ENDOR results indicate a common mechanism of framework substitution in which Mn(II) and Fe(III) are replacing Al (or Mg). Moreover, the spectra serve as a probe for the differences in the local environment and bonding topology of these substituted framework sites. A qualitative interpretation of the ³¹p ENDOR data is provided, based on relevant crystallographic information, and the ¹H ENDOR signals are partially attributed to the interactions with the templates occluded in the zeotype cages. To further relate the isotropic ³¹p hyperfine couplings to structural properties, DFT methods were employed for cluster model optimizations and hyperfine coupling constants calculations. Geometry optimizations of substituted rings, derived from the SOD and AEL framework structures, indicate considerable distortions of the coordination environment of framework Mn as compared to Al. A systematic study of the hyperfine interactions of a series of model structures containing tetrahedral and octahedrai Mn(II) show that both Mn-O bond lengths and Mn-O-P bond angles contribute significantly to the variation in the isotropic and anisotropic ³¹p hyperfine coupling.

The electron spin-echo envelope modulation (ESEEM) technique was used to investigate the formation mechanism of the mesoporous material MCM-41. The spin-probes 4-(N, N-dimethyl-N-hexadecyl)ammonium-2, 2, 6, 6, -tetramethyl piperidine-oxyl iodide (CAT16) and 5-doxyl stearic acid (5DSA) were introduced into the surfactant (cetyltrimethylammonium bromide, CTAB) solution in minute amounts followed by the addition of a base and a silica source to initiate the reaction. The reaction was then quenched at different times by rapid insertion into liquid nitrogen. The preservation of the micellar structure upon freezing was proved by a series of ESEEM measurements carried out on 5DSA in CTAB solutions of various concentrations, which showed that the ¹⁴N modulation depth was sensitive to the transition from spherical to cylindrical micelles. Variations in the immediate environment of the spin-probes occurring during the room temperature formation of MCM-41 were followed by tracing the ²H modulation depth k(²H) induced by α-d₂-CTAB molecules and D₂O. For both spin-probes, k(²H) of α-d₂-CTAB decreased throughout the reaction, whereas k(₂H) of D₂O showed a small increase. In all cases, the time evolution of k(²H) revealed two stages: one that lasted for the first ∼12 min, during which most changes have occurred, followed by a second, longer one with mild changes. The reduction of k(²H) of α-d₂-CTAB in the case of 5DSA was assigned to its displacement toward the organic core, driven by charge repulsion between negatively charged silicate oligomers at the interface and the negative polar head of 5DSA. Considering the different position of the nitroxide spin label in CAT16 and 5DSA with respect to the α-position in the CTAB molecules, the decrease in k(²H) for CAT16 was attributed to an upward displacement, and a protrusion into the soft silica layer, driven by steric consideration and charge attraction. The slight increase in k(²H) due to D₂O shows that the silica layer formed in the room temperature synthesis is water rich such that the density of water and OH groups in the vicinity of the spin-probes increases. The majority of the water, however, is easily removed just by filtering the solid formed and drying at room temperature. Finally, evidence for the rearrangement of surfactant molecules and the increase of the aggregate size during the first stage of the reaction was obtained from changes in the echo decay time.

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(II) complexes with L-histidine and DL-histidine-α-d,β-d₂ were prepared in H₂O and in D₂O, and orientation-selective W-band ¹H and ²H pulsed ENDOR spectra of these complexes were recorded at 4.5 K. These measurements lead to the unambiguous assignment of the signals of the H_α, H_β, imidazole H_ε, and the exchangeable amino, H_am, protons. The ¹⁴N superhyperfine splitting observed in the X-band EPR spectrum and the presence of only one type of H_α and H_β protons in the W-band ENDOR spectra show that the complex is a symmetric bis complex. Its g11 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 g11. From the anisotropic part of the hyperfine interaction of H_α and H_β and applying the point-dipole approximation, a structural model was derived. An unexpectedly large isotropic hyperfine coupling, 10.9 MHz, was found for H_α. In contrast, H_α 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_α can serve as a signature for a histamine coordination where both the amino and imino nitrogens of the same molecule bind to the Cu(II), forming a six-membered chelating ring. Unlike H_α the hyperfine coupling of H_ε is not as sensitive to the presence of a coordinated amino nitrogen of the same histidine molecule.

The effect of axial ligand mutation on the Cu_A site in the recombinant water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase ba₃ has been investigated. The weak methionine ligand was replaced by glutamate and glutamine which are stronger ligands. Two constructs, M160T0 and M160T9, that differ in the length of the peptide were prepared. M160T0 is the original soluble fragment construct of cytochrome ba₃ that encodes 135 amino acids of subunit II, omitting the transmembrane helix that anchors the domain in the membrane. In M160T9 nine C-terminal amino acids are missing, including one histidine. The latter has been used to reduce the amount of a secondary T2 copper which is most probably coordinated to a surface histidine in M160T0. The changes in the spin density in the Cu_A site, as manifested by the hyperfine couplings of the weakly and strongly coupled nitrogens, and of the cysteine β-protons, were followed using a combination of advanced EPR techniques. X-band (∼9 GHz) electron-spin - echo envelope modulation (ESEEM) and two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy were employed to measure the weakly coupled ¹⁴N nuclei, and X- and W-band (95 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy for probing the strongly coupled ¹⁴N nuclei and the β-protons. The high field measurements were extremely useful as they allowed us to resolve the T2 and Cu_A signals in the g_⊥ region and gave ¹H ENDOR spectra free of overlapping ¹⁴N signals. The effects of the M160Q and M160E mutations were: (i) increase in A_II(^63,65Cu), (ii) larger hyperfine coupling of the weakly coupled backbone nitrogen of C153, (iii) reduction in the isotropic hyperfine interaction, a_iso, of some of the β-protons making them more similar, (iv) the a_iso value of one of the remote nitrogens of the histidine residues is decreased, thus distinguishing the two histidines, and finally, (v) the symmetry of the g-tensor remained axial. These effects were associated with an increase in the Cu - Cu distance and subtle changes in the geometry of the Cu₂S₂ core which are consistent with the electronic structural model of Gamelin et al.

Two different approaches for assigning electron nuclear double resonance (ENDOR) signals to their respective M_s manifolds by a controlled generation of asymmetric ENDOR spectra, are described and applied to a number of systems. This assignment then allows a straightforward determination of the sign of the hyperfine coupling. Both approaches rely on a high thermal polarization that is easily achieved at high fields and low temperatures. For high-spin systems, such as S = 5/2 the assignment is afforded by the selective inversion of the | - 3/2> → | - 1/2> electron paramagnetic resonance (EPR) transition which is highly populated as compared to its symmetric counterpart, the |1/2> → |3/2> EPR transition, and therefore is easily identified. For S = 1/2 the determination of the sign of the hyperfine coupling becomes possible when the cross-and nuclear-spin relaxation rates are much slower than the electron-spin relaxation rate and variable mixing time pulse ENDOR is used to measure the spectrum. Under these conditions the signals of the M_s = 1/2 (α) manifold become negative when the mixing time is on order of the electron-spin relaxation time, whereas those of the M_s = - 1/2 (β) manifold remain positive. Under partial saturation of the nuclear transitions and short mixing time the opposite behavior is observed. Pulse W-band ¹H ENDOR experiments demonstrating these approaches were applied and the signs of the hyperfine couplings of the water ligands in Mn(H₂O)²⁺₆ the H_α and H_β, histidine protons in the Cu(histidine)₂ complex, the imidazole protons in Cu(imidazole)²⁺₄ and the cysteine β-protons in nitrous oxide reductase were determined.

Pulsed electron nuclear double-resonance (ENDOR) spectroscopy at W- and X-band frequencies has been employed to characterize the structure of NO adsorption sites involving sodium cations in zeolite NaA. The principal values of the sodium hyperfine and nuclear quadrupole coupling tensors as well as the orientation of their principal axes system with respect to the g tensor coordinate frame of the Na⁺ - NO adsorption complex could be determined by orientation-selective ENDOR spectroscopy. Such orientation-selective experiments benefit especially from the high spectral resolution at W-band frequencies. Furthermore, the sodium ENDOR spectrum is drastically simplified at high frequencies where the limit of weak hyperfine couplings is fulfilled. The dipolar sodium hyperfine coupling tensor reveals a bent structure of the formed adsorption complexes and gives access to the bond distance between the NO molecule and the cations. The Na nuclear quadrupole data indicate that the adsorption complexes are preferentially formed with the sodium ions at the six-membered rings of the NaA zeolite structure. An analysis of the sodium and nitrogen hyperfine coupling data shows that 96% of the unpaired electron spin density in the Na⁺ - NO adsorption complex is localized in the nitrogen and oxygen 2pπ orbitals of the NO ligand molecule.

A two-dimensional (2D) experiment that correlates electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) frequencies, useful for unraveling and assigning ENDOR and ESEEM spectra from different paramagnetic centers with overlapping EPR spectra, is presented. The pulse sequence employed is similar to the Davies ENDOR experiment with the exception that the two-pulse echo detection is replaced by a stimulated echo detection in order to enhance the resolution in the ESEEM dimension. The two-dimensional data set is acquired by measuring the ENDOR spectrum as a function of the time interval T between the last two microwave pulses of the stimulated echo detection scheme. This produces a series of ENDOR spectra with amplitudes that are modulated with T. Fourier transformation (FT) with respect to T then generates a 2D spectrum with cross peaks connecting spectral lines of the ESEEM and ENDOR spectra that belong to the same paramagnetic center. Projections along the vertical and horizontal axes give the three-pulse FT-ESEEM and ENDOR spectra, respectively. The feasibility of the experiment was tested by simulating 2D ENDOR-ESEEM correlation spectra of a system consisting of an electron spin (S = 1/2) coupled to two nuclei (I₁ = I₂ = 1/2), taking into account / experimental conditions such as pulse durations and off-resonance irradiation frequencies. The experiment is demonstrated on a single crystal of Cu²⁺ doped L-histidine (Cu-His), containing two symmetrically related Cu²⁺ sites that at an arbitrary orientation exhibit overlapping ESEEM and ENDOR spectra. While the ESEEM spectrum is relatively simple and arises primarily from one weakly coupled ¹⁴N, the ENDOR spectrum is very crowded due to contributions from two nonequivalent nitrogens, two chlorides, and a relatively large number of protons. The simple ESEEM projection of the 2D ENDOR-ESEEM correlation spectrum is then used to disentangle the ENDOR spectrum and resolve two sets of lines corresponding to the different sites.

The formation mechanism of the hexagonal mesoporous material MCM-41, prepared with tetraethyl-orthosilicon (TEOS) and cetyl-trimethylammonium chloride (bromide) (CTAC/B), was investigated through the motional characteristics of the spin probe 5-doxyl stearic acid (5DSA). Electron spin echo envelope modulation (ESEEM) experiments, carried out on the final product, showed that the spin probe is incorporated into the organic part and the nitroxide radical is located near the organic-inorganic interface. The EPR spectra of 5DSA were measured in situ during the formation of MCM-41 at 298 K. The spectra were analyzed by computer simulations that provide the time evolution of the rotational diffusion rates, R_⊥ and R_∥, and of the ordering potential. As the reaction progresses, the spin probe, which reflects the behavior of the surfactant molecules, experiences an increasing order parameter, S, while its rotational diffusion rates decrease. From the time evolution of these parameters two stages were distinguished. During the first, which lasts about 12 min, S, R_⊥ and R_∥ change rapidly whereas during the second, which lasts about 1 h, R_⊥ and R_∥ remain essentially constant while S exhibits a mild increase. The fast stage is assigned to the onset of orientational ordering and silicate condensation, which occur simultaneously, while the slow process reflects the "hardening" of the silica wall.

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 ≃ 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 ≃ 1.97 signal, with similar saturation characteristics was found in the AO: azide (1: 7) sample. The g 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear: azide complex.

The incorporation of Mn(II) into framework sites in the aluminophosphate zeotype AlPO₄-20, an analog of sodalite, has been investigated using pulsed electron nuclear double resonance (ENDOR) spectroscopy at 95 GHz. The field sweep echo-detected EPR spectrum showed the presence of a single Mn(II) site with a ⁵⁵Mn hyperfine coupling of 8.7 mT. ENDOR spectra, recorded using the Mims and Davies sequences, consist of an ²⁷Al signal at the Larmor frequency and a ³¹P doublet corresponding to a hyperfine coupling of 8 MHz. The symmetry of the doublet about the ³¹P Larmor frequency indicates that it originates from the Ms = ± 1/2 manifolds and that the interaction is primarily isotropic. The relatively large ³¹P hyperfine interaction and the weak interaction with ²⁷Al provide unique and direct evidence for Mn(II) substitution of framework Al. X-band electron spin echo envelope modulation (ESEEM) measurements showed only signals at the ¹H, ¹⁴N, ²⁷Al, and ³¹P Larmor frequencies. The first two are due to weak dipolar interaction with the template molecules, while the others reflect interactions with the framework.

A two-dimensional experiment, termed DONUT-HYSCORE (double nuclear coherence transfer hyperfine sublevel correlation) designed to obtain correlations between nuclear frequencies belonging to the same electron spin manifold is presented. The sequence employed is π/2-τ₁-π/2-t₁-π-τ₂- π-t₂-π/2τ₁-echo, and the echo is measured as a function of t₁ and t₂ whereas τ₁ and τ₂ are held constant. It is complementary to the standard HYSCORE experiment which generates correlations between nuclear frequencies belonging to different M(s) manifolds and is particularly useful for ¹⁴N nuclei. The experiment is first demonstrated on a single crystal of copper- doped l-histidine hydrochloride monohydrate where the modulations are induced by a single ¹⁴N nucleus, the remote nitrogen in the imidazole group. HYSCORE and DONUTHY-SCORE experiments were carried out on two crystal orientations. In the first, one Cu²⁺ site contributes to the echo and all six nuclear frequencies together with the expected correlation were observed. In the second, 12 frequencies corresponding to two Cu²⁺ ions at different crystallographic sites appeared and all expected correlations were detected as well. This rather trivial example demonstrates that the DONUT-HYSCORE pulse sequence indeed generates correlations within the M(s) manifolds. The value of the DONUT-HYSCORE experiment is demonstrated on a frozen solution of a vanadyl complex with a bis-hydroxamate ion binder (VO-RL515). The modulations in this complex arise from the two InN nuclei in the hydroxamate groups, and orientation-selective three-pulse ESEEM (electron spin-echo envelope modulation) spectra showed a number of well-resolved peaks. An unambiguous assignment of all peaks and their orientation dependences could not be achieved through HYSCORE alone because at certain orientations frequencies of one of the M(s) manifolds were absent or overlapped with those of the other manifold. The application of the DONUT-HYSCORE experiment provided new correlations that led to the complete assignment of the ESEEM frequencies, thus paving the way for future systematic spectral simulations for the determination of the best-fit Hamiltonian parameters. This example shows that, in the case that the HYSCORE experiment cannot distinguish between two sets of frequencies belonging to the same M(s) manifold in different centers (or orientations) because signals from the other manifold are missing or overlapping, the DONUT-HYSCORE becomes most valuable.

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

Two-dimensional (2D) pulsed EPR spectroscopy was applied to study the copper ligands in azurins from Pseudomonas aeruginosa, Az(pae), and Alcaligenes species NCIIB 11015, Az(asp), in frozen solutions. While a high-resolution three-dimensional crystal structure is available for Az(pae), only a low-resolution structure has been reported for Az(asp). Az(pae) was studied in the pH range 3.9-7.0 and Az(asp) at a pH of 4.8. Measurements were performed at 9 GHz which is usually within the cancellation condition for the remote nitrogen of imidazole ligands. The main technique was the hyperfine sublevel correlation (HYSCORE) technique. At all pH values investigated the 2D HYSCORE spectra of Az(pae) showed correlations between the nuclear frequencies corresponding to the nuclear quadrupole resonance (NQR) frequencies of the remote nitrogens of the imidazole ligands and the double quantum frequency. The spectra showed additional well-resolved cross peaks which indicate correlations between the NQR frequencies of a weakly coupled amide nitrogen and the corresponding double quantum frequency. This confirms earlier detection and assignment of the electron spin-echo envelope modulation (ESEEM) frequencies of this nitrogen which were based on ESEEM measurements of the H117G mutant (Coremans et al. Chem. Phys. Lett. 1995, 235, 202). The 2D spectra of Az(asp) were similar to those of Az(pae) showing that a third weakly coupled nitrogen is present in this species as well. HYSCORE spectra of a frozen solution of ascorbate oxidase exhibited only signals corresponding to the remote nitrogens of the imidazole. Comparing these spectra with those of the azurins and correlating the results with the available crystal structures of ascorbate oxidase and Az(pae) suggest that the third nitrogen in Az(pae) is the amide nitrogen of His-46, coupled to the copper via the carbonyl group of Gly-45. This further implies that also in azurin from Az(asp), the precise 3D structure of which is not yet a

Orientation selective and multiple frequency ESEEM experiments on substrate free cytochrome P450cam (CP450cam) with ¹⁷O-enriched water are reported. The ¹⁷O ESEEM frequencies were obtained from Fourier transformation of the ratio of ESEEM waveforms of CP450cam with enriched water and CP450cam with non-enriched water. Numerical simulations were carried out to determine the isotropic and the anisotropic hyperfine interactions and the quadrupole interaction of the ¹⁷O. From the magnitude (e²qQ/h = 6.6 MHz) and asymmetry (η = 0.95) of the ¹⁷O 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 ¹⁷O quadrupole tensor, the orientation of the water molecule was found to be confined (within ±10°) with respect to the FeN directions within the heme plane. Two possible sets of isotropic and anisotropic components of the ¹⁷O 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 ¹⁷O 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.

The binding of Cu(II) to a lipophilic bis-hydroxamate binder, RL252, and its parent RL239, was investigated by pulsed EPR. The binders have two arms, each terminated with a hydroxamate group which serves as a donor. The major difference between RL252 and RL239 is the absence of the leucine amino acid bridge in RL239. Orientation-selective electron spin echo envelope modulation (ESEEM) experiments were carried out at 8.45 and 9.15 GHz. The spectra obtained were exceptionally well resolved and indicated that the cancellation condition, which requires that the hyperfine coupling is approximately twice the nuclear Larmor frequency, is met at 89 GHz. The spectra of both CuRL252 and CuRL239 showed the nuclear quadrupole resonance (NQR) frequencies, ν₀, ν, and ν₊, of the ¹⁴N of the hydroxamate groups at 1.61.7, 2.152.25, and 3.83.9 MHz. A peak corresponding to the double quantum transition in the \u201cnoncanceled manifold\u201d, ν_DQ, was observed at 55.2 MHz, and at some magnetic fields a peak corresponding to a single quantum transition, ν_SQ, and its combination harmonic appeared as well. The assignment of all the ESEEM frequencies was achieved by the application of the two-dimensional hyperfine sublevel correlation (HYSCORE) experiment. Following the assignment, simulations of the orientation-selective ESEEM spectra were performed, yielding the magnitude and orientation of the quadrupole and hyperfine tensors of the coupled nitrogens. While only one best fit set of quadrupolar parameters was found, two such sets were obtained for the hyperfine interaction. Analysis of the orientation of the quadrupole tensor with respect to the g-tensor showed that in both CuRL252 and CuRL239 the binding site is close to coplanar. Very subtle differences in the spin Hamiltonian parameters were observed between CuRL252 and CuRL239. The ¹⁴N quadrupolar parameters and the anisotropic hyperfine component were slightly larger in Cu-RL239. The relatively small ¹⁴N hyperfine coupling is attributed to a node in the molecular orbital, occupied by the unpaired electron, at the nitrogen.

This chapter focuses on the synthesis of mesoporous manganosilicate materials Mn-M41 S, having hexagonal (Mn-MCM-41), cubic (Mn-MCM-48) and lamellar (Mn-MCM-L) structures, at a low surfactant/silica ratio (0.12), and present a preliminary account on the location of the Mn²⁺ cations as obtained from Q-band EPR spectroscopy. It shows that the addition of Mn ions induces the formation of the cubic phase also at a low surfactant/Si ratio (0.12), and that by the variation the base content of the gel, one can control the structure formed. Mesoporous manganosilicate molecular sieve, Mn-M41 S, 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 dynamic processes in tetrahydrofuran (THF) solutions of 2,5-di-tert-butyl-p-benzoquinone^-, Na⁺ (DtBPBQ^-, Na⁺) ion-pairs, obtained by reduction with a sodium mirror, have been studied by two-dimensional (2D) exchange Fourier transform (FT) EPR spectroscopy. Measurements were made at room temperature (17-20 °C) on solutions with radical concentrations ranging from 4.5 × 10^-5 to 3.5 × 10^-3 M and with mixing times varying from 0.3 to 6 μs. Analysis of the EPR spectra indicates the presence of two types of DtBPBQ^-, Na⁺ ion-pairs, which are labeled A and B. In both species there is intramolecular Na⁺ hopping, but while in ion-pair A the process is slow and suitable for monitoring by the 2D exchange method, in ion-pair B it is much faster and results in a selective smearing of some of the hyperfine lines. The origin of the latter is tentatively ascribed to complexation with OH^- generated by water impurity. In the 2D spectra characteristic cross peaks due to Na⁺ hopping and Heisenberg exchange (HE) appear. Analysis of these spectra provides information about the intramolecular Na⁺ hopping rate in ion-pair A, [formula omitted], as well as on the HE rate constants, k_ii, of the various radicals. At room temperature these are [formula omitted], and k_BB = (1.0 ± 0.2) × 10⁸ s^-1 moh¹. These results also provide information on the longitudinal relaxation rates of the overall magnetization of both radicals. These depend on the total radical concentration and within experimental accuracy are the same for both radicals. The mechanism for this process is tentatively ascribed to electron-electron (radical-radical) dipolar interaction, and its rate is compared with calculations based on the point dipole approximation. The present work demonstrates the power of the 2D exchange EPR method in elucidating mechanisms of dynamic processes and determining kinetic parameters, in particular when several such processes occur simultaneously.

A variety of magnetic resonance experiments were performed to investigate the local environment of Mn in MnAPSO-44. MnAPSO-44 is a member of the aluminophosphate molecular sieves family and has a chabazite-like structure. Two samples with different Mn contents (Mn/(P + Al + Si) = 0.9 and 0.07 atom %) in their as-synthesized, calcined hydrated and dehydrated forms were studied. The ³¹P, ²⁹Si, and ²⁷A1 MAS NMR spectra are similar to those of the corresponding SAPO-44 samples showing only one type of TO₄ tetrahedra. In the hydrated sample an ²⁷Al signal at −13 ppm, characteristic of octahedral Al, appears as well due to water coordination. EPR spectra were measured at X- and Q-band. The Mn(II) in the as-synthesized and calcined samples showed hyperfine splittings of 85 and 93 G, respectively, the latter being characteristic of octahedral environment. Dehydration at 400 °C reduced the hyperfine constant to 65 G, indicating a change to tetrahedral coordination upon water removal. The nuclei in the immediate surrounding of the Mn were probed by the electron spin echo envelope modulation (ESEEM) technique. Both ³¹P and ²⁷Al modulations were observed. The EPR and ESEEM results are interpreted in terms of Mn incorporation into tetrahedral framework sites for the sample with the low Mn content. The spatial distribution of the Mn throughout the sample was investigated by the \u201c2+1\u201d electron spin echo (ESE) experiment. It was found that only about 15% of the Mn(II) is homogeneously distributed and contributes to the echo signal.

The blue oxidases, laccase and ascorbate oxidase, contain three spectroscopically distinct copper binding sites, two of which are EPR detectable in the oxidized Cu(II) state, called type 1 (T1) and type 2 (T2). The three dimensional structure of ascorbate oxidase has recently been determined (Messerschmidt A. et al.: J. Mol. Biol. 206, 513 (1989)) while that of laccase has not. We have therefore carried out comparative electron spin echo envelope modulation (ESEEM) measurements on ascorbate oxidase, laccase and laccase in which T1 Cu(II) was substituted with Hg(II) in order to obtain structural information about the copper sites in laccase. The ESEEM results clearly show that there are as many histidines in laccase as in ascorbate oxidase, i.e., at least two at each site. Orientation selective ESEEM experiments showed that in the T1 site in both enzymes the two remote (uncoordinated) nitrogens are magnetically inequivalent and have different hyperfine interactions. Furthermore, the isotropic hyperfine constants of both remote nitrogens in laccase T1 are larger than those in ascorbate oxidase T1. In laccase T2 two remote nitrogens show similar hyperfine couplings and the modulation depth is significantly deeper than in ascorbate oxidase. Finally, it is suggested that the difference between the NQR frequencies of the remote nitrogens in T1 and T2 in both oxidases can be attributed to the alkyl group of the side chain being adjacent to the bound imidazole in T1 and to the remote nitrogen in T2. This is in accordance with the known X-ray structure of ascorbate oxidase.

Electron spin echo envelope modulation (ESEEM) induced by ²⁷Al 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 ²⁷Al nuclear quadrupole interaction which is often large and unknown. In order to obtain a better understanding of the various spectral features of the ²⁷Al 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 Cu ²⁺ 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 e ²qQ/h 2qQ/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.

In an attempt to provide models for copper binding in proteins, symmetric and nonsymmetric chiral ligands designed to bind copper in a controlled geometry were synthesized. These ligands are assembled from trifunctional anchors extended by donors containing amino acids such as histidine and methionine. The copper coordination of these complexes was studied by orientation selective electron spin echo envelope modulation (ESEEM) experiments. The three-pulse FT-ESEEM spectra consist of peaks at 0.7, 1.4, 2.1, and 3.0 MHz, which positions are practically independent on the resonant magnetic field, and a peak at about 4 MHz, which shows significant field dependence. The relative intensities of all lines vary with the resonant field. These lines are typical for the remote nitrogen in the imidazole ring. By using computer simulations of the FT-ESEEM spectra recorded at the various resonant magnetic fields along the powder pattern and taking into account the selected excited orientations, the ¹⁴N isotropic and anisotropic hyperfine interactions were determined. The simulations also gave the relative orientation of the ¹⁴N hyperfine and quadrupole tensor principal axes with respect to the g tensor principal axis. From these parameters it was concluded that in the complexes consisting of three histidyl residues all three imidazoles are coordinated to the copper, not in a coplanar structure but in a propeller-like arrangement. No other nitrogens, such as the pivotal nitrogen, were found to be coordinated to the copper.

Multinuclear NMR measurements were performed on several samples of AlPO₄-5: samples that are as synthesized, calcined and rehydrated, dehydrated and those obtained after adsorption of methanol and ammonia. The dynamics of the adsorbates was followed by ²H NMR, which was correlated with the changes the framework undergoes upon adsorption, as manifested by the ³¹P and ²⁷Al MAS NMR spectrum. Water adsorption up to 20% of the total AlPO₄-5 capacity did not affect the ²⁷Al and ³¹P spectra, and the ²H NMR spectrum indicates the presence of rather mobile water molecules. When the samples are completely hydrated, the Al spectrum shows the presence of octahedral Al, the ³¹P line is significantly broadened, and the ²H NMR shows the appearance of "bound" water molecules undergoing a local motion, which could be a π flip about the DOD bisector. These molecules are assigned to water coordinated to the octahedral Al and the flip is about the Al-O axis. The ²H NMR spectrum of adsorbed ND₃ indicates the presence of two types of molecules, just as in the case of water, with the bound molecules undergoing a rotation about the C₃ axis, along the Al-N axis. In this case three types of Al were detected, tetrahedral, octahedral, and pentacoordinated. While water and ammonia behaved similarly, methanol adsorption did not produce octahedral Al and the methanol molecules were found to freely reorient within the AlPO₄-5 channels. ³¹P T₂ measurements attributed the changes in the ³¹P line width to inhomogeneous broadening due to dispersion of chemical shifts. This dispersion was assigned to the changes in the Al neighbors.