Publications

The distance distributions between two site-specifically anchored spin labels in a protein, measured by pulsed electron-electron double resonance (PELDOR or DEER), provide rich sources of structural and conformational restraints on the proteins or their complexes. The rigid connection of the nitroxide spin label to the protein improves the accuracy and precision of distance measurement. We report a new spin labelling approach by formation of thioester bond between nitroxide (NO) spin label, NOAI (NO spin labels activated by either acetylimidazole), and a protein thiol, and this spin label method has demonstrated high performance in DEER distance measurement on proteins. The results showed that NOAI has shorter connection to the protein ligation site than 2, 2, 5, 5-tetramethyl-pyrroline-1-oxyl methanethiosulfonate (MTSL) and 3-maleimido-proxyl (M-Prox) in the respective protein conjugate and produces narrower distance distributions for the tested proteins including ubiquitin (Ub), immunoglobulin-binding β1 domain of streptococcal protein G (GB1), and second mitochondria-derived activator of caspases (Smac). The NOAI protein conjugate connected by a thioester bond is resistant to reducing reagent and offers high-fidelity DEER distance measurements in cell lysates.

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.

Heat shock protein 90 (Hsp90) serves as a crucial regulator of cellular proteostasis by stabilizing and regulating the activity of numerous substrates, many of which are oncogenic proteins. Therefore, Hsp90 is a drug target for cancer therapy. Hsp90 comprises three structural domains, a highly conserved amino-terminal domain (NTD), a middle domain (MD), and a carboxyl-terminal domain (CTD). The CTD is responsible for protein dimerization, is crucial for Hsp90's activity, and has therefore been targeted for inhibiting Hsp90. Here we addressed the question of whether the CTD dimerization in Hsp90, in the absence of bound nucleotides, is modulated by allosteric effects from the other domains. We studied full length (FL) and isolated CTD (isoC) yeast Hsp90 spin-labeled with a Gd(III) tag by double electron-electron resonance measurements to track structural differences and to determine the apparent dissociation constant (K_d). We found the distance distributions for both the FL and isoC to be similar, indicating that the removal of the NTD and MD does not significantly affect the structure of the CTD dimer. The low-temperature double electron-electron resonance-derived K_d values, as well as those obtained at room temperature using microscale thermophoresis and native mass spectrometry, collectively suggested the presence of some allosteric effects from the NTDs and MDs on the CTD dimerization stability in the apo state. This was evidenced by a moderate increase in the K_d for the isoC compared with the FL mutants. Our results reveal a fine regulation of the CTD dimerization by allosteric modulation, which may have implications for drug targeting strategies in cancer therapy.

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.

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

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.

In this article we describe the electron-electron double resonance (ELDOR)-detected NMR (EDNMR) method. It is one in a number of the hyperfine spectroscopy techniques, where the aim is to detect magnetic nuclei coupled with the unpaired electron and to determine their hyperfine couplings, which are too small to be resolved in the EPR spectrum. EDNMR does so by recording the NMR spectrum of these coupled nuclei via the electron spin signal; it complements the electon-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) methods described in other articles. We first introduce the experiment and give an intuitive explanation of its spin-physics mechanism, along with a few examples. We then discuss its pros and cons by comparing it with ENDOR and ESEEM and continue by briefly presenting ways to simulate the spectra. We end with a description of more advanced two-dimensional EDNMR experiments.

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

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.

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.

Dynamic nuclear polarization is typically explained either using microscopic systems, such as in the solid effect and cross effect mechanisms, or using the macroscopic formalism of spin temperature which assumes that the state of the electrons can be described using temperature coefficients, giving rise to the thermal mixing mechanism. The distinction between these mechanisms is typically made by measuring the DNP spectrum-i.e. the nuclear enhancement profile as a function of irradiation frequency. In particular, we have previously used the solid effect and cross effect mechanisms to explain temperature dependent DNP spectra. Our past analysis has however neglected the effect of depolarization of the electrons resulting from the microwave (MW) irradiation. In this work we concentrate on this electron depolarization process and perform electron-electron double resonance (ELDOR) experiments on TEMPOL and trityl frozen solutions, using a 3.34 Tesla magnet and at 2.7-30 K, in order to measure the state of the electron polarization during DNP. The experiments indicate that a significant part of the EPR line is affected by the irradiation due to spectral diffusion. Using a theoretical framework based on rate equations for the polarizations of the different electron spin packets and for those of the nuclei we simulated the various ELDOR line-shapes and reproduced the MW frequency and irradiation time dependence. The obtained electron polarization distribution cannot be described using temperature coefficients as required by the classical thermal mixing mechanism, and therefore the DNP mechanism cannot be described by thermal mixing. Instead, the theoretical framework presented here for the analysis of the ELDOR data forms a basis for future interpretation of DNP spectra in combination with EPR measurements.

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.

Distance measurements using double electron-electron resonance (DEER) and Gd³⁺ chelates for spin labels (GdSL) have been shown to be an attractive alternative to nitroxide spin labels at W-band (95 GHz). The maximal distance that can be accessed by DEER measurements and the sensitivity of such measurements strongly depends on the phase relaxation of Gd³⁺ chelates in frozen, glassy solutions. In this work, we explore the phase relaxation of Gd³⁺-DOTA as a representative of GdSL in temperature and concentration ranges typically used for W-band DEER measurements. We observed that in addition to the usual mechanisms of phase relaxation known for nitroxide based spin labels, GdSL are subjected to an additional phase relaxation mechanism that features an increase in the relaxation rate from the center to the periphery of the EPR spectrum. Since the EPR spectrum of GdSL is the sum of subspectra of the individual EPR transitions, we attribute this field dependence to transition dependent phase relaxation. Using simulations of the EPR spectra and its decomposition into the individual transition subspectra, we isolated the phase relaxation of each transition and found that its rate increases with |m_s|. We suggest that this mechanism is due to transient zero field splitting (tZFS), where its magnitude and correlation time are scaled down and distributed as compared with similar situations in liquids. This tZFS induced phase relaxation mechanism becomes dominant (or at least significant) when all other well-known phase relaxation mechanisms, such as spectral diffusion caused by nuclear spin diffusion, instantaneous and electron spin spectral diffusion, are significantly suppressed by matrix deuteration and low concentration, and when the temperature is sufficiently low to disable spin lattice interaction as a source of phase relaxation.

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.

The ¹³C solid state Dynamic Nuclear Polarization (DNP) mechanism using trityl radicals (OX63) as polarizers was investigated in the temperature range of 10-60 K. The solutions used were 6 M ¹³C urea in DMSO/H ₂O (50% v/v) with 15 mM and 30 mM OX63. The measurements were carried out at ∼3.5 T, which corresponds to Larmor frequencies of 95 GHz and 36 MHz for the OX63 and the ¹³C nuclei, respectively. Measurements of the ¹³C signal intensity as a function of the microwave (MW) irradiation frequency yielded ¹³C DNP spectra with temperature dependent lineshapes for both samples. The maximum enhancement for the 30 mM sample was reached at 40 K, while that of the 15 mM sample at 20-30 K. Furthermore, the lineshapes observed showed that both the cross effect (CE) and the solid effect (SE) DNP mechanisms are active in this temperature range and that their relative contribution is temperature dependent. Simulations of the spectra with the relative contributions of the CE and SE mechanisms as a fit parameter revealed that for both samples the CE contribution decreases with decreasing temperature while the SE contribution increases. In addition, for the 15 mM sample the contributions of the two mechanisms are comparable from 20 K to 60 K while for the 30 mM the CE dominates in this range, as expected from the higher concentration. The steep decrease of the CE contribution towards low temperatures is however unexpected. The temperature dependence of the OX63 longitudinal relaxation, DNP buildup times and ¹³C spin lattice relaxation times did not reveal any obvious correlation with the DNP temperature dependence. A similar behavior of the CE and SE mechanism was observed for ¹H DNP with the nitroxide radical TEMPOL as a polarizer. This suggests that this effect is a general phenomenon involving a temperature dependent competition between the CE and SE mechanisms, the source of which is, however, still unknown.

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.

In nitrite reductase (cd(1) NIR), the c-heme mediates electron transfer to the catalytic d(1)-heme where nitrite (NO2-) is reduced to nitric oxide (NO). An interesting feature of this enzyme is the relative lability of the reaction product NO bound to the d(1)-heme. Marked differences in the c- to d(1)-heme electron-transfer rates were reported for cd(1) NIRs from different sources, such as Pseudomonas stutzeri (P. stutzeri) and Pseudomonas aeruginosa (P. aeruginosa). The three-dimensional structure of the P. aeruginosa enzyme has been determined, but that of the P. stutzeri enzyme is still unknown. The difference in electron transfer rates prompted a comparison of the structural properties of the d(1)-heme pocket of P. stutzeri cell NIR with those of the P. aeruginosa wild type enzyme (WT) and its Y10F using their nitrosyl d(1)-heme complexes. We applied high field pulse electron paramagnetic resonance (EPR) techniques that detect nuclear spins in the close environment of the spin bearing Fe(II)-NO entity. We observed similarities in the rhombic g-tensor and detected a proximal histidine ligand with N-14 hyperfine and quadrupole interactions also similar to those of P. aeruginosa WT and Y10F mutant complexes. In contrast, we also observed significant differences in the H-bond network involving the NO ligand and a larger solvent accessibility for P. stutzeri attributed to the absence of this tyrosine residue. For P. aeruginosa, cd, NIR domain swapping allows Tyr(10) to become H-bonded to the bound NO substrate. These findings support a previous suggestion that the large difference in the c- to d(1)-heme electron transfer rates between the two enzymes is related to solvent accessibility of their d(1)-heme pockets.

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.

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.

We present high field DEER (double electron-electron resonance) distance measurements using Gd³⁺ (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd³⁺ spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd³⁺ was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd³⁺ labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd³⁺ labeled peptide we explored the effect of the solution molar ratio of Gd ³⁺ and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd³⁺]/[Tag] ratio is crucial because excess Gd³⁺ masked the DEER effect and too little Gd³⁺ resulted in the formation of Gd³⁺-tag₂ complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd³⁺ concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd³⁺ ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.

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.

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.

Ascorbate oxidase contains two paramagnetic Cu(ii) binding sites, type 1 (T1) and type 2 (T2) and in both sites the Cu(ii) is coordinated to histidine residues. We use several pulse EPR techniques at high field (95 GHz) to determine ligand ¹H and ¹⁴N hyperfine couplings in the two sites and identify the T1 signals by a new triple resonance correlation technique named THYCOS.

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.

Pulse double electron-electron spin resonance (DEER) measurements were applied to characterize the distribution and average number of guest-molecules (in the form of spin-probes) in Pluronic P123 micelles. Two types of spin-probes were used, one of which is a spin-labeled P123 (P123-NO), which is similar to the micelles constituent molecules, and the other is spin-labeled Brij56 (Brij56-NO) which is significantly different. Qualitative information regarding the relative location of the spin-labels within the micelles was obtained from the isotropic hyperfine coupling and the correlation times, determined from continuous wave EPR measurements. In addition, complementary small angle X-ray scattering (SAXS) measurements on the P123 micellar solutions, with and without the spin-probes, were carried out for an independent determination of the size of the core and corona of the micelles and to ensure that the spin-probes do not alter the size or shape of the micelles. Two approaches were used for the analysis of the DEER data. The first is model free, which is based on the determination of the leveling off value of the DEER kinetics. This provided good estimates of the number of radicals per micelle (low limit) which, together with the known concentration of the P123 molecules, gave the aggregation number of the P123 micelles. In addition, it provided an average distance between radicals which is within the range expected from the micelles' size determined by SAXS. The second approach was to analyze the full kinetic form which is model dependent. This analysis showed that both spin-labels are not homogeneously distributed in either a sphere or a spherical shell, and that large distances are preferred. This analysis yielded a slightly larger occupation volume within the micelle for P123-NO than for Brij56-NO, consistent with their chemical character.

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.

Double electron - electron resonance (DEER)is employed to explore the evolution of solution structures during the formation of the bicontinuous cubic (Ia3̄d) mesoporous material KIT-6. We focused on the early stages of the reaction, where micellar structures are not well-resolved in the micrographs of cryogenic transmission electron microscopy (cryo-TEM). KIT-6 is synthesized with Pluronic P123 block copolymers, PEO₂₀PPO_70- PEO ₂₀, as a template. Initially, the aqueous solution, held at 40 °C, consists of Pluronic P123 micelles, which are characterized by a hydrophilic corona, comprising the poly(ethylene oxide) (PEO) blocks, and a hydrophobic core, consisting of the poly(propylene oxide) (PPO) block. The variations in the volume of the hydrophobic core of the micelles as a consequence of the addition of the silica source (TEOS, tetraethoxyorthosilane) were evaluated using a hydrophobic spin-probe, 4-hydroxy-tempobenzoate (4HTB), which is localized in the hydrophobic core of the micelles. The measurements were carried out on solutions that were freeze - quenched at different times after the addition of TEOS (tetraethoxyorthosilane). New details on the formation of KIT-6 were obtained, revealing swelling of the hydrophobic core during the first ∼10 min of the reaction followed by a contraction, close to the original size. The swelling was attributed to penetration of the TEOS and its hydrolysis products into the micelles.

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.

(100-x) mol % B₂O₃ x mol % Me₂O (Me=Li,Na,K) glasses, exposed to γ-⁶⁰ Co irradiation to produce paramagnetic states, were characterized by W -band (95 GHz) pulse electron-nuclear double resonance (ENDOR) spectroscopy in order to characterize local structures occurring in the range of compositions between x=16 and x=25 at which the "boron oxide" anomaly occurs. The high resolution of nuclear frequencies allowed resolving the ⁷Li and ¹¹B ENDOR lines. In the samples with x=16 and x=20 glasses, ¹¹B hyperfine couplings of 16, 24, and 36 MHz were observed and attributed to the tetraborate, triborate, and boron oxygen hole center (BOHC) structures, respectively. The x=25 samples showed hyperfine couplings of 15 MHz for the tetraborate and 36 MHz for BOHC. Density functional theory (DFT) calculations predicted for these structures negative hyperfine couplings, which were confirmed by W -band ENDOR. This suggests that a spin polarization mechanism accounts for the negative hyperfine structure splitting.

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.

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn²⁺ and the other, S2, by Ca²⁺. ⁵⁵Mn electron-nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, ∼3.5 T) to determine the ⁵⁵Mn nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the ⁵⁵Mn ENDOR rotation patterns showed that two chemically inequivalent Mn²⁺ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e²Qq/h, are significantly different; 10.7 ± 0.6 MHz for Mn_A²⁺ and only -2.7 ± 0.6 MHz for Mn_B²⁺. The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for Mn_A²⁺ and -263.5 MHz for Mn_B²⁺. The principal z-axis for Mn_A²⁺ is not aligned with any of the Mn-ligand directions, but is 25° off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of Mn_B²⁺ the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90° with respect to Mn_A. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn²⁺, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e²Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally.

The catalytic activity of the tertiary stabilized hammerhead ribozyme (tsHHRz) is by three orders of magnitude higher than the one of the long-known minimal construct (mHHRz). This gives rise to the question whether the single high-affinity manganese(II) binding site present in both ribozymes is located closer to the cleavage site and the transition state in the tsHHRz than in the mHHRz, which would make a direct involvement of this metal(II) ion in the bond-breaking step more likely. Here, we used W-band ³¹P-Davies-ENDOR (electron-nuclear double resonance) to complement earlier reported ¹⁴N-ESEEM/HYSCORE (electron spin echo envelope modulation/hyperfine sublevel correlation) studies. The ³¹P-ENDOR spectrum of the mHHRz revealed a doublet with a splitting of 8.4(±0.5) MHz but unresolved hyperfine anisotropy. Such a large splitting indicates an inner-sphere coordination of a phosphate backbone group with a significant amount of spin density on the phosphorous nucleus. This is in good agreement with the ³¹P isotropic hyperfine constant, A_iso(³¹P), of +7.8 MHz obtained by density functional theory calculations on the structure of the Mn²⁺ binding site as found in crystals of the same ribozyme. This supports the idea that the structure and location of the binding site in the mHHRz is in frozen buffer similar to that found in the crystal. Since the W-band ENDOR spectrum of the tsHHRz also shows a ³¹P splitting of 8.4(±0.5) MHz, the local structures of both binding sites appear to be similar, which agrees with the coincidence of the ¹⁴N data. An involvement of the high-affinity Mn²⁺ ion in the catalytic step seems therefore unlikely.

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.

We put forward a theoretical model for the morphological transitions of templated mesoporous materials. These materials consist of a mixture of surfactant molecules and inorganic compounds which evolve dynamically upon mixing to form different morphologies depending on the composition and conditions at which mixing occurs. Our theoretical analysis is based on the assumption that adsorption of the inorganic compounds onto mesoscopic assemblies of surfactant molecules changes the effective interactions between the surfactant molecules, consequently lowering the spontaneous curvature of the surfactant layer and inducing morphological changes in the system. On the basis of a mean field phase diagram, we are able to follow the trajectories of the system starting with different initial conditions, and predict the final morphology of the product. In a typical scenario, the reduction in the spontaneous curvature leads first to a smooth transition from compact spherical micelles to elongated wormlike micelles. In the second stage, the layer of inorganic material coating the micelles gives rise to attractive inter-micellar interactions that eventually induce a collapse of the system into a closely packed hexagonal array of coated cylinders. Other pathways may lead to different structures including disordered bicontinuous and ordered cubic phases. The model is in good qualitative agreement with experimental observations.

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.

The complexes of copper with histidine exhibit a wide variety of coordination modes in aqueous solution. This stems from the three potential coordination sites of the histidine molecule and the existence of mono- and bis-complexes. The present work concentrates on the determination of the carboxylate binding mode, via the ¹³C hyperfine coupling of the carboxyl, in a number of copper complexes in frozen solutions. These are then used as references for the determination of the coordination mode of two zeolite encapsulated complexes. The ¹³C hyperfine coupling (sign and magnitude) was determined by a variety of advanced pulsed EPR and electron-nuclear double resonance (ENDOR) techniques carried out at conventional and high magnetic fields. These showed that while the carboxyl ¹³C isotropic hyperfine coupling of an equatorially coordinated carboxylate is negative with a magnitude of 3-4 MHz, that of a free carboxylate is small (≃1 MHz) and positive. To rationalize the experimentally determined ligand hyperfine couplings (¹H and ¹³C) and further understand their dependence on the coordination mode and degree of protonation, density functional theory (DFT) calculations were carried out on a number of model complexes, representing the different Cu-histidine complexes studied experimentally. The exchange-correlation functional used for the calculation of the EPR parameters was B3LYP with triple-ζ plus polarization (TZP) quality basis sets. While the polarization agreement between the magnitudes of the calculated and experimental values varied among the various nuclei, sometimes exhibiting deviations of up to 40%, an excellent agreement was found for the sign prediction. This shows the unique advantage of combining high field ENDOR, by which the sign of the hyperfine can often be determined, with DFT predictions for structure determination.

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 effect of dispersion and ensembling mode of chromium oxide nanocrystals in bulk xerogels and aerogels on their performance as well as efficiency of high surface area chromia aerogel as catalysts support were investigated in complete ethylacetate (EA) oxidation. Two series of chromia catalysts with a structure of α-Cr₂O₃ (7-110m ²g^-1) and α-CrOOH (230-735m²g ^-1) were tested in EA complete oxidation as a model reaction for VOC combustion. For α-Cr₂O₃, the specific activity increased 3.3 times with decreasing the crystal diameter from ~100 to 13nm. For α-CrOOH materials, where the surface area was determined by the different packing mode of primary nanoparticles of the same size (3-5nm), the similar specific rate constants were measured with all the tested samples. The activity of the chromia aerogel (α-CrOOH, 630m²g^-1) was four times higher compared with 0.5% Pt/Al₂O₃ and 30 times higher relative to 30% Cr₂O₃/SiO₂. Oxidative treatment (O₂) at elevated temperatures converts both phases to CrO_2, in case of α-Cr₂O₃ - only in the crystals surface layer. In both reduced and oxidized states, high concentration of surface oxygen vacancies were detected in α-Cr₂O ₃ and α-CrOOH catalysts. A redox cycle Cr(III)[]OH↔Cr(IV) [O]O which determines the catalysts performance at the surface of both types of bulk chromia materials was proposed. Promotion of high surface area chromia aerogel with Pt, Au, Mn and Ce increased its activity in EA complete oxidation by a factor of 1.25-2.7. Addition of Pt, Mn to ceria-promoted chromia aerogel has a significant effect, yielding a high specific rate constant. Promotion with Ce- and Mn-additives improved the efficiency of redox cycle in Cr-aerogel and increased the concentration of surface oxygen vacancies.

Continuous-wave and pulse electron paramagnetic resonance (EPR) as well as electron-nuclear double resonance (ENDOR) techniques are applied for determination of the electronic and geometric structure of copper(II) complexes with terpyridine and related ligands that are relevant in the context of supramolecular chemistry. The results are analysed in conjunction with density functional theory computations and are compared to the crystal structure of the bis(terpyridyl) copper(II) complex in the limit of static Jahn-Teller distortion (R. Allmann, W. Henke and D. Reinen, Inorg. Chem. 1978, 17, 378, ref. 6). The static structure in disordered environments is subject to some strain in both the g-values and copper-ligand distances, but otherwise is rather similar to the structure in crystals. The formation of coordination oligomers is indicated by broadening of the lineshape and a decrease in the transverse relaxation time at low fields which are both related to exchange coupling between copper centres. At high fields of approximately 3 T and a temperature of 15 K, the transverse relaxation rate is governed by modulation of the g-values induced by small-amplitude libration along the Jahn-Teller active mode. A study of the dynamics in a temperature range from below the glass transition temperature to above the melting point of ethanol by CW EPR reveals that the complex is a sensitive probe for matrix dynamics, which detects dynamic heterogeneities and the transition from the structural glass to the crystalline phase. Jahn-Teller dynamics is completely unfrozen only on melting of the matrix.

Keywords: Biochemistry & Molecular Biology; Chemistry, Inorganic & Nuclear

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.

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.

High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn²⁺ ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H₂O and in D₂O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of ∼3.4 T allowed us to assign the ENDOR signals to their respective M_s manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn²⁺ ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein (Deacon et al. J. Chem. Soc., Faraday Trans. 1997, 93(24), 4305-4312).

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.

The incorporation of Mn(II) into the mesoporous material MCM-41, synthesized under acidic conditions with [Si]/[H⁺] in the range of 0.1 - 0.4 was investigated. Tetraethyl-orthosilicon (TEOS) was used as the silica source and cetyltrimethylammonium chloride or bromide (CTAC/CTAB) as the structure-directing agent. The Mn(II) sites in the final product were characterized by high-field (W-band, 95 GHz) pulsed EPR and electron - nuclear double resonance (ENDOR) spectroscopies that provided highly resolved EPR spectra and detailed information concerning the Mn(II) coordination sphere. These measurements were complemented with X-band continuous wave (CW) EPR and electron-spin - echo envelope modulation (ESEEM) spectroscopies. In addition, the bulk properties of the final products were characterized by X-ray diffraction and ²⁹Si MAS NMR, while in situ X-band CW EPR measurements on reaction mixtures containing the spin probe 5-doxyl stearic acid (5DSA) were carded out to follow the reaction kinetics and the degree of silica condensation. These bulk properties were then correlated with the formation of the different Mn(II) sites. The final product consists of a mixture of two hexagonal phases (d = 38, 43 Å), the relative amounts of which depend on the [Si]/[H⁺] in the starting gel. Two Mn(II) sites, which exhibit unresolved overlapping signal in the X-band EPR spectra, were easily distinguished in the low-temperature field-sweep (FS) echo-detected (ED) W-band spectra due to their significantly different ⁵⁵Mn hyperfine couplings. One Mn(II) site, a, is characterized by a hyperfine splitting of 97 G, and the second, b, by 82 G. The relative amount of b increases with increased acidity. Site a is assigned to a hexa-coordinated Mn(II) with water ligands that is anchored to the internal pore surface of the silica either by one coordination site or through hydrogen bond(s). The Mn(II) in site b is in a distorted tetrahedral coordination, located within the first few layers of the silica wall. When the water content of the final products increases, site b assumes characteristics that are very similar to site a, suggesting that the silica wall is 'soft' and not fully condensed. The NMR and in situ EPR measurements show that incomplete TEOS hydrolysis and slow silica polymerization, which occurs for [Si]/[H⁺] > 0.1, favors the formation of site a, whereas complete hydrolysis and fast polymerization, generating enough acidic Si - OH groups, favors the formation of site b. The incomplete hydrolysis also accounts for the generation of two hexagonal phases.

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.

The incorporation of Fe(III), during the synthesis, into aluminosilicate sodalite (FeSOD) and aluminophosphate sodalite, AIPO₄-20 (FAPO), was investigated by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques at X- and W-band. Specifically, the effect of the framework composition and the presence of occluded template molecules (tetramethyl ammonium hydroxide, TMAOH) in the β cages on the distribution of the Fe(III) species was explored. The X-band CW EPR spectrum of FAPO shows the existence of two types of species, one with a large (g ≈ 6.3, 4) and the other with a small (g ≈ 2) zero field splitting (ZFS) interaction. These species were also found in FeSOD synthesized with TMAOH. The X-band field-sweep echo-detected (FS-ED) EPR spectrum shows contributions only from the Fe(III) species in the more symmetric environment (g ≈ 2). The other was not detected due to fast relaxation. This spectrum is very broad and suffers from distortions due to the nuclear modulation effect. In contrast, the W-band FS-ED EPR spectrum of the same species was significantly narrower and free from distortions. Analysis of the temperature dependence of the width and relative intensity of the peak corresponding to the |-1/2] → |+1/2] EPR transition shows that the g ≈ 2 signal arises from a number of Fe(III) species with a distribution of ZFS parameters. Calcination significantly reduces the ZFS parameter, D, suggesting that the distortions of the T sites are due to specific interactions with the template. Electron spin echo envelope modulation (ESEEM) experiments shows the presence of weak dipolar interaction between Fe(III) and template ¹⁴N and ¹H template nuclei, as well as framework ²⁷Al and ³¹P nuclei. This indicates that the species characterized by small ZFS are well dispersed and are located within the inner structure of the zeolite. These g ≈ 2 species are most probably Fe(III) in framework sites. A small fraction that occupies highly asymmetric sites (g ≈ 6.3, 4), situated at 'defect' framework or extraframework sites, and some Fe(II) produced due to the reduction of Fe(III) by the organic template (detected by Mössbauer spectroscopy), were found as well. The possible presence of some extraframework Fe(III) with a g ≈ 2 signal cannot be excluded.

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

The role of the Cu(II) in the catalytic oxidation of CO over Cu/SnO₂ with low Cu(II) content was studied by continuous wave EPR, electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopes. Three methods were employed for introducing the copper: (i) by coprecipitation, (ii) impregnation onto SnO₂ gel and (iii) impregnation onto calcined SnO₂. Two types of Cu(II) species were identified in these calcined Cu/SnO₂ materials. Those belonging to the first type, termed B and C, exhibit highly resolved EPR spectra with well defined EPR parameters and are located within the bulk of the oxide. The other group comprises a distribution of surface Cu(II) species with unresolved EPR features and are referred to as S. While the latter were readily reduced by CO the former required long exposures at high temperatures (> 673 K). The specific interactions of the different Cu(II) species with CO were investigated through the determination of the ¹³C hyperfine coupling of enriched ¹³CO. The ESEEM spectra of calcined samples, generated either by coprecipitation or impregnation, show after the adsorption of CO signals at the Larmor frequencies of ^117,119Sn and ¹³C and at twice these Larmor frequencies. Although these signals indicate that ^117,119Sn and ¹³C are in the close vicinity of Cu(II), they cannot provide the hyperfine couplings of these nuclei. This problem was overcome by the application of the HYSCORE experiment. The 2D HYSCORE spectra show well resolved cross peaks which provide the hyperfine interaction of these nuclei. Simulations of the HYSCORE spectra yield for ^117,119Sn an isotropic hyperfine constant, a_iso, of ±4.0 MHz and an anisotropic component, T_⊥, of ±2.0 MHz. Pulsed ENDOR spectra also showed ^117,119Sn signals which agree with the above values. The ¹³C cross peaks yield a_iso = ±1.0 MHz and T_⊥ = ±2.0 MHz. Similar C cross peaks were observed in spectra of calcined Cu/SnO₂ after the adsorption of CO₂. Based on the same hyperfine couplings in the samples exposed to ¹³CO and ¹³CO₂ the signals were assigned to surface carbonate species generated by part of the Cu(II) S type species rather then by species B and the role of the Cu(II) in the oxidation process is discussed.

The binding of VO²⁺to chiral dihydroxamate binders facilitates the transport of VO²⁺through the cell membrane into the cell interior, where it was shown to simulate glucose metabolism (Shechter, Y.; Shisheva, A.; Lazar, R.; Libman, J.; Shanzer, A. Biochemistry 1992, 31, 2063). The unique structure of the binders relies on a modular dipodal topology which generates different binding cavities. The coordination of VO²⁺to two homologues of these ligands. RL261 and RL239, having different dipod arms but identical donor groups, was investigated by orientation selective electron spin echo envelope modulation (ESEEM) spectroscopy. Relatively deep modulations were observed for the ¹⁴N nuclei in the hydroxamate groups in both complexes owing to the fulfillment of the cancellation condition at ~9 GHz. The Fourier transform (FT) ESEEM spectra showed four peaks, three corresponding to the nuclear quadrupole resonance (NQR) lines, v_o, v_-, and v₊, and one to the overtone, 2v_m. In VO-RL261 the NQR and the 2v_mpeaks appear at 1.75, 2.15, 3.9, and 6.0 MHz, respectively, whereas in VO-RL239 they are at 1.75, 2.05, 3.9, and 6.2 MHz, respectively. From the positions of these peaks the ¹⁴N quadrupole coupling constant, |e²qQ/h|, the asymmetry parameter, η, and the isotropic hyperfine constant |a_iso|, were estimated to be 4.0, 0.87, and 2.5 MHz, respectively, for VO-RL261 and 3.8, 0.91, and 2.8 MHz, respectively, for VO-RL239. The unique orientation dependence of the v₊peak, which practically disappeared when the field was set to A_||(⁵¹V), indicates that the principal axis of the quadrupole tensor, z', is either parallel or perpendicular to the VO axis. In order to obtain more accurate values of the above parameters and to determine the anisotropic hyperfine component, a_⊥, as well as the orientations of the hyperfine and quadrupole tensors with respect to the VO axis, a series of simulations were carried out. The best fit parameters showed that a_⊥is rather large (0.6-0.7 MHz) and cannot be neglected and that a_isois smaller than expected, i.e., 1.6-1.8 MHz. We also obtained that z is to a good approximation parallel to the VO axis indicating that the two hydroxamate planes are perpendicular to the VO axis in both complexes. Two possible structures, one with a C₂symmetry, trans configuration, and one with a σ_xzsymmetry, cis configuration, were considered in the simulations and the latter was found to agree better with the experimental results. The slight differences in the parameters obtained for VO-RL261 and VO-RL239 are attributed to electronic effects induced by the different groups bounded to the hydroxamate carbonyl.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Pharmacology & Pharmacy

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

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.

Electron spin echo studies have been carried out for a series of x-doxylstearic acid (x-DSA, where x = 5, 10, 16) spin probes in three different sets of samples of a five-component microemulsion (water, toluene, 1-butanol, sodium dodecyl sulfate (SDS), and sodium chloride) selectively deuterated in water, toluene, or 1-butanol. The sequence Winsor I, III, II was obtained by changing the salinity from 0.943 × 10^-2 to 3.31 × 10^-2 mole ratio, corresponding to 3% and 10% (w/w) in the nondeuterated system. Modulation effects due to interactions of the nitroxide group with water deuteriums, perdeuterated toluene, or 1-butanol have been measured as a function of x and of the brine concentration, S_M. The average probe location and probe conformation in the microemulsions as a function of x are reported. The probe conformations support the presence of oil-in-water and water-in-oil "structures" in Winsor I and Winsor II microemulsions, respectively. The comparison of the deuterium modulation depth magnitude between Winsor I microemulsions and the SDS/water/1-butanol/sodium chloride micellar system, selectively deuterated in the water or butanol, shows at a molecular level that the Winsor I microemulsions and the SDS micellar system have very similar 1-butanol distritubtions and the same water penetration at the interface. It is also found that toluene penetrates the SDS layer at the microemulsion interface up to 4-5 methylenes from the SDS polar headgroup. The Winsor I to Winsor III phase transition is associated with a consistent decrease of the toluene penetration into the SDS interfacial layer and probably with a variation of the SDS alkyl chains packing and/or conformation. Furthermore, the "oil" region, the "water" region, and the "interfacial" region seem to be well separated for all the microemulsions studied. The butanol is mainly located at the water side of the interface. It is also shown that the interface of bicontinuous microemulsions is little affected by S_M variation.

The immediate environment of Mn(II) in MnAlPO5 after synthesis, calcination, hydration, and dehydration has been investigated by both conventional ESR spectroscopy and by several electron spin-echo (ESE) methods. Several samples with Mn(II) contents between Mn/P = 0.05 and 0.5 atom % were synthesized, and samples of impregnated Mn-AlPO5 and exchanged Mn-SAPO5 were used as references. The X-band and Q-band ESR spectra of all as-synthesized and calcined MnAlPO5 samples and of impregnated Mn-AlO5 are characteristic of Mn(II) in a slightly distorted octahedral symmetry with an hyperfine coupling constant of 90 G and a zero-field splitting parameter of 140 G. The spatial distribution of the Mn(II) cations was investigated by the "2 + 1" ESE experiment. A random homogeneous distribution of isolated Mn(II) cations was found only in the sample with the lowest Mn(II) content. In all other samples regions with enriched Mn(II) concentrations, where most of the Mn(II) are located, were found to exist. Dehydration causes the migration of the Mn(II) cations toward each other, and consequently the spin exchange interaction increases, resulting in the averaging of the hyperfine interaction. Electron spin-echo envelope modulation (ESEEM) experiments, which detect only the isolated Mn(II) species, showed that the isolated Mn(II) cations interact through weak dipolar interactions with an average of 6 ³¹P nuclei at a distance of 5 Å. This does not agree with a model of Mn(II) substituting for framework Al. Significant changes in the Al modulation after calcination and hydration indicated that this treatment results in a change in the Mn(II) location. Mn(II) in calcined and hydrated MnAlPO5 and in impregnated Mn-AlPO5 is coordinated to three or four framework oxygens, and the remaining ligands are water molecules and/or hydroxyl groups. All the above observations lead to the conclusion that the majority of the Mn(II) in MnAlPO5 does not occupy framework sites but is probably coordinated to the external surface.