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.

Double electron-electron resonance (DEER) measures distances between spin labels attached at well-defined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work, we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd(III) and delivered into human HeLa cells by electro-poration (EP) and hypotonic swelling (HS). Gd(III)-Gd(III) DEER measurements were carried out on cells frozen after different incubation times, following delivery to test the stability of the spin label inside the cell. For both delivery methods, it was possible to derive distance distributions up to 12 h after delivery, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, only one mutant reported a significant change in the distance distribution with time and only for HS delivery. On the basis of in vitro exchange experiments with Mn(II) and comparison with the same mutant labeled with BrPSPy-DO3MA-Gd(III) and considering the presence of Mn(II) in the cell, we hypothesized that the change occurred as a consequence of partial Gd(III)/Mn(II) exchange with endogenous Mn(II). These experiments also showed that the relative Gd(III)/Mn(II) binding affinity depends on the labeling site in the protein, which accounts for the lack of change with the other mutants delivered under HS conditions. We conclude that 4PS-PyMTA-Gd(III) is a good spin label for in-cell DEER for delivery by EP, but caution should be taken when HS is used.

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.

Here, we present an integrated experimental and theoretical study of ¹H dynamic nuclear polarization (DNP) of a frozen aqueous glass containing free radicals at 7 T, under static conditions and at temperatures ranging between 4 and 20 K. The DNP studies were performed with a home-built 200 GHz quasi-optics microwave bridge, powered by a tunable solid-state diode source. DNP using monochromatic and continuous wave (cw) irradiation applied to the electron paramagnetic resonance (EPR) spectrum of the radicals induces the transfer of polarization from the electron spins to the surrounding nuclei of the solvent and solutes in the frozen aqueous glass. In our systematic experimental study, the DNP enhanced ¹H signals are monitored as a function of microwave frequency, microwave power, radical concentration, and temperature, and are interpreted with the help of electron spin-lattice relaxation times, experimental MW irradiation parameters, and the electron spectral diffusion (eSD) model introduced previously. This comprehensive experimental DNP study with mono-nitroxide radical spin probes was accompanied with theoretical calculations. Our results consistently demonstrate that eSD effects can be significant at 7 T under static DNP conditions, and can be systematically modulated by experimental conditions.

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.

Mn²⁺ chelating tags for Mn²⁺-Mn²⁺ distance measurements by pulse EPR spectroscopy were developed. They feature a stable C-S conjugation to the protein, high reactivity towards cysteine thiols and short and rigid linkers that can be used in distance measurements with high resolution under reductive conditions. Double electron-electron resonance measurements at 95 GHz on ubiquitin labeled with these tags showed the expected narrow distance distribution.

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

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

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

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

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

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

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

Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd³⁺ chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd³⁺ central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 μs in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd³⁺- nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.

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.

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

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

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

cd₁ nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d₁-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d₁-heme to avoid enzyme inhibition by the formation of ferrous-nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d₁-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d₁-heme NO complex from Pseudomonas aeruginosa cd₁ NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr₁₀, His₃₂₇, and His₃₆₉), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d₁-heme complex of cd₁ NIR forms H-bonds with Tyr₁₀ and His₃₆₉, whereas the second conserved histidine, His₃₂₇, appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr₁₀ is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His₃₆₉ is closer to the NO, whereas mutation of both distal histidines displaces Tyr ₁₀, removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.

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

The binding of NO to reduced myoglobin in solution results in the formation of two paramagnetic nitrosyl myoglobin (MbNO) complexes: one with a rhombic g-factor and the other with an axial one, referred to as the R- and A-forms. In spite of past extensive studies of MbNO by crystallography, spectroscopy and quantum chemical calculations it is still not clear what factors determine the appearance of the two forms. In this work we applied a combination of state of the art quantum chemical calculations and high field pulsed EPR spectroscopy (W-band, 3.4 T/95 GHz) to further characterize the two forms. Specifically, we have used ¹H and ²H electron-nuclear double resonance (ENDOR) spectroscopy to identify and characterize the H-bond to the NO, and hyperfine sub-level correlation (HYSCORE) spectroscopy to determine the hyperfine and quadrupole interactions of the Fe(ii) coordinated ¹⁴N of the proximal histidine ¹⁴N_His93. The calculations employed quantum mechanics (QM), particularly density functional theory (DFT) methods in combination with molecular mechanics (MM) force-field to model the protein environment. Through QM/MM calculations of the EPR parameters we have explored their dependence on several geometrical factors of the Fe-NO bond and found those that reproduce the best experimental results. The spread of the W-band EPR spectrum of MbNO due to the g-anisotropy is large and there is a significant part of the spectrum where the R-form is the sole contributor. This allowed us to resolve some new characteristics of the R-form: (i) a NO-H hydrogen bond has been detected and characterized and through QM/MM calculations has been unambiguously assigned to ^ɛ2H_His64. (ii) The complete hyperfine and quadrupole interactions of ¹⁴N_His93 have been determined and correlated with structural parameters again using QM/MM calculations. The agreement between the experimental results and calculations varied between excellent and good, depending on the EPR parameter in question. As for the more elusive A-form, the results only suggest that it does have a ¹⁴N_His93 ligand with a hyperfine comparable to that of the R-form and it has less hydrogen bonding interaction with His₆₄. The calculations also established the orientation of the principal g-values, finding that they are closely related to the orientation of the NO bond. This information is essential for deriving structural information from the experimental orientation selective HYSCORE and ENDOR data.

Mesoporous alumina with a narrow pore size distribution can be synthesized by hydrolysis of aluminum alkyl ethers in an organic solvent in the presence of an ionic or nonionic surfactant. However, the pores are not ordered and the role of the surfactant as a possible template is not clear as it is in the synthesis of the mesoporous silica of the M41S family and of the SBA-15, 16 type materials. In this work we use continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy, in combination with electron spin-echo envelope modulation (ESEEM) measurements, to provide experimental evidence for the interaction between the surfactant and the alumina species during synthesis. This is achieved by using spin-labeled analogs of the surfactants and following their interaction with ²⁷Al at different stages of the alumina formation. The results show that in sec-butanol solutions the surfactants do not form micellar aggregates. Instead, the anionic surfactant, lauric acid, has a strong tendency to bind to alumina precursors, hydrolysis products of the aluminum alkyl ethers in the starting solution, and remains bound until the final product. Here the surfactant decorates the surface of the alumina particles with an average extended configuration with the carboxylate being the closest to the alumina surface. The interaction with aluminum increases during precipitation as the density of the alumina increases. By contrast, the nonionic polyethyleneoxide type surfactants: Tergitol 15-S-12 and Pluronics P123 and L64 seem to remain unbound even in the as-synthesized precipitates. In this last case, EPR spectra of a small, hydrophobic spin probe have shown that solvent evaporation by room temperature drying brings about the formation of liquid-like "organic zones" confined in the alumina structure, which presumably are at the origin of the pores.

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

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

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

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

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.

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

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

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.

Davies electron-nuclear double resonance spectra can exhibit strong asymmetries for long mixing times, short repetition times, and large thermal polarizations. These asymmetries can be used to determine nuclear relaxation rates in paramagnetic systems. Measurements of frozen solutions of copper(L-histidine)₂ reveal a strong field dependence of the relaxation rates of the protons in the histidine ligand, increasing from low (g_∥) to high (g_⊥) field. It is shown that this can be attributed to a concentration-dependent T_1e-driven relaxation process involving strongly mixed states of three spins: the histidine proton, the Cu(II) electron spin of the same complex, and another distant electron spin with a resonance frequency differing from the spectrometer frequency approximately by the proton Larmor frequency. The protons relax more efficiently in the g_⊥ region, since the number of distant electrons able to participate in this relaxation mechanism is higher than in the g _∥ region. Analytical expressions for the associated nuclear polarization decay rate T_een^-1 are developed and Monte Carlo simulations are carried out, reproducing both the field and the concentration dependences of the nuclear relaxation.

An EPR spectrum of as synthesized [G.A. Tsigdinos, C.J. Hallada, Inorg. Chem. 7 (1968) 437-441], orange colored, H₅PV₂Mo₁₀O₄₀ polyoxometalate showed the presence of a reduced vanadium(IV) addenda atom. Surprisingly, further ³¹P ENDOR (electron-nuclear double resonance) measurements indicated the absence of a phosphorous heteroatom leading to the suggestion that H₅V^VV^IVMo₁₁O₄₀ exists as a previously unrecognized impurity in the typically synthesized H₅PV₂Mo₁₀O₄₀ compound. H_5/4PV^VO₄V^IV/VMo₁₁O₃₆ was then synthesized in low yield (0.8 mol%) by omitting the addition of phosphate in a typical H₅PV₂Mo₁₀O₄₀ preparation. The molecular formulation and structure was supported by X-ray crystallography, infrared and mass spectrometry. Further use of EPR/ENDOR/ESEEM (electron-spin echo envelope modulation) allowed the formulation of [V^VV^IVMo₁₁O₄₀]^5- as [V^VO₄V^IVMo₁₁O₃₆]^5-. Accordingly, the polyoxometalate has a multiscripts(VO, 4, mml:none(), mml:none(), 3 -) heteroatom core with 11 molybdenum addenda and one VO²⁺ moiety at the polyoxometalate surface. The redox potential and the catalytic activity of the new vanadomolybdate polyoxometalate compound were essentially identical to the often-studied H₅PV₂Mo₁₀O₄₀ polyoxometalate isomeric mixture.

The determination of the details of the spatial and electronic structure of functional sites (centers) in any system, be it in materials chemistry or in biology, is the first step towards understanding their function. When such sites happen to be paramagnetic in any point of their activity cycle, the tool box offered by a variety of high resolution electron paramagnetic resonance (EPR) spectroscopic techniques becomes very attractive for their characterization. This tool box has been considerably expanded by the developments in high field (HF) EPR in general, and HF electron nuclear double resonance (ENDOR), in particular. These have led to numerous new applications in the fields of biology, physics, chemistry and materials sciences. This overview focuses specifically on recent applications of pulsed HF ENDOR spectroscopy to microporous materials, such as zeotype materials, presenting the new opportunities it offers. First, a brief description of the theoretical basis required for the analysis of the HF ENDOR spectrum is given, followed by a description of the pulsed techniques used to record spectra and assign the signals, along with a brief presentation of the required instrumentation. Next, specific applications are given, including transition metal ions and complexes exchanged into zeolite cages, transition metal substitution into frameworks of zeolites, aluminophosphate molecular sieves, and silicious mesoporous materials, the interaction of NO with Lewis sites in zeolite cages and trapped S ^-₃ . We end with a discussion of the advantages and the shortcomings of the method and conclude with a future outlook.

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

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.

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

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

The 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.

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

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.

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

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

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.

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.

Orientation selective and multiple frequency ESEEM experiments on substrate free cytochrome P450cam (CP450cam) with ¹⁷O-enriched water are reported. The ¹⁷O ESEEM frequencies were obtained from Fourier transformation of the ratio of ESEEM waveforms of CP450cam with enriched water and CP450cam with non-enriched water. Numerical simulations were carried out to determine the isotropic and the anisotropic hyperfine interactions and the quadrupole interaction of the ¹⁷O. From the magnitude (e²qQ/h = 6.6 MHz) and asymmetry (η = 0.95) of the ¹⁷O quadrupole interaction, we conclude that the distal axial ligand of Fe³⁺ in the CP450 heme is a water molecule. Moreover, from the orientation of the ¹⁷O quadrupole tensor, the orientation of the water molecule was found to be confined (within ±10°) with respect to the FeN directions within the heme plane. Two possible sets of isotropic and anisotropic components of the ¹⁷O hyperfine interaction, (±2.6, ±0.3 MHz) and (±0.4, ±1.8 MHz), were found to satisfactorily reproduce the experimental results. Both sets indicate very small ¹⁷O hyperfine couplings which is consistent with the unpaired electron residing predominately in the d_yz orbital of the Fe³⁺. Ligand field models for each set are presented.

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

This chapter focuses on the synthesis of mesoporous manganosilicate materials Mn-M41 S, having hexagonal (Mn-MCM-41), cubic (Mn-MCM-48) and lamellar (Mn-MCM-L) structures, at a low surfactant/silica ratio (0.12), and present a preliminary account on the location of the Mn²⁺ cations as obtained from Q-band EPR spectroscopy. It shows that the addition of Mn ions induces the formation of the cubic phase also at a low surfactant/Si ratio (0.12), and that by the variation the base content of the gel, one can control the structure formed. Mesoporous manganosilicate molecular sieve, Mn-M41 S, having hexagonal (Mn-MCM-41), cubic (Mn-MCM-48), lamellar (Mn-MCM-L) structure, were synthesized in low surfactant/Si ratio (0.12) and characterized by X-ray powder diffraction, transmission electron microscopy (TEM), themogravimetric analysis (TGA) and electron paramagnetic resonance (EPR). The phase transformations trends: hexagonal ∼ lamellar ∼ cubic -∼ hexagonal or hexagonal hexagonal, lamellar mixture ∼ cubic ∼ lamellar were observed by variations of the base or acid content of the gel or reaction temperature respectively.

The dynamic processes in tetrahydrofuran (THF) solutions of 2,5-di-tert-butyl-p-benzoquinone^-, Na⁺ (DtBPBQ^-, Na⁺) ion-pairs, obtained by reduction with a sodium mirror, have been studied by two-dimensional (2D) exchange Fourier transform (FT) EPR spectroscopy. Measurements were made at room temperature (17-20 °C) on solutions with radical concentrations ranging from 4.5 × 10^-5 to 3.5 × 10^-3 M and with mixing times varying from 0.3 to 6 μs. Analysis of the EPR spectra indicates the presence of two types of DtBPBQ^-, Na⁺ ion-pairs, which are labeled A and B. In both species there is intramolecular Na⁺ hopping, but while in ion-pair A the process is slow and suitable for monitoring by the 2D exchange method, in ion-pair B it is much faster and results in a selective smearing of some of the hyperfine lines. The origin of the latter is tentatively ascribed to complexation with OH^- generated by water impurity. In the 2D spectra characteristic cross peaks due to Na⁺ hopping and Heisenberg exchange (HE) appear. Analysis of these spectra provides information about the intramolecular Na⁺ hopping rate in ion-pair A, [formula omitted], as well as on the HE rate constants, k_ii, of the various radicals. At room temperature these are [formula omitted], and k_BB = (1.0 ± 0.2) × 10⁸ s^-1 moh¹. These results also provide information on the longitudinal relaxation rates of the overall magnetization of both radicals. These depend on the total radical concentration and within experimental accuracy are the same for both radicals. The mechanism for this process is tentatively ascribed to electron-electron (radical-radical) dipolar interaction, and its rate is compared with calculations based on the point dipole approximation. The present work demonstrates the power of the 2D exchange EPR method in elucidating mechanisms of dynamic processes and determining kinetic parameters, in particular when several such processes occur simultaneously.

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

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

Electron spin echo methods were applied to characterize the immediate environment and the spatial distribution of paramagnetic transition metal cations in microporous solids. Electron spin echo envelope modulation (ESEEM) induced by framework ²⁷A1 nuclei was used to investigate the different sites of Cu²⁺ in zeolites X, Y and A. All Cu²⁺ species obtained after complete dehydration showed relatively large isotropic hyperfine interactions with framework Al. This is indicative of a firm interaction of the cation with the framework oxygens. Orientation selective experiments provided additional information concerning the arrangement of the ²⁷A1 nuclei around the Cu²⁺ and led to site identification. Furthermore, such measurements separated the contributions of distant and near Al nuclei to the modulation. The immediate environment of Mn²⁺ in MnAlPO-5 where the Mn²⁺ was incorporated during synthesis as compared to post synthesis introduction via impregnation was investigated. ESEEM induced by framework ³¹P and ²⁷A1 nuclei was used to investigate the location of the Mn²⁺ cations with respect to these framework elements and the spatial distribution of the Mn²⁺ centers throughout the structure was studied by the \u201d2+1\u201d ESE pulse sequence. All the experimental results obtained by these methods led to the conclusion that in MnAlPO-5 the Mn^a+ is not situated in the framework but is rather bounded to the external surface via terminal framework oxygens.

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.

Deuterium Nuclear Magnetic Resonance studies in discotic mesophases are reviewed. The studies include NMR measurements on neat compounds as well as deuterated probe molecules dissolved in the mesophase. The discotic compounds discussed include derivatives of benzene, triphenylene and truxene which exhibit a variety of mesophases. Particular emphasis is placed on comparing axially symmetric and biaxial phases, and on analyzing the various orientational order parameters required to characterize their NMR spectra. The conformation of side chains as derived from the deuterium resonance of the methylene and methyl deuterons is also reviewed. Finally, the properties of discotic mesophases as ordering solvents for solute probe molecules are discussed.

Deuterium, oxygen-17 and sodium-23 NMR measurements in the mesophase region of the disodiumchromoglycate (DSCG)-water system are reported. The results indicate that, depending on the temperature and concentration of the DSCG, at least three different mesophases can exist in the system: At temperature below minus 4 degree C a relatively highly ordered smectic-like phase exists (phase III) which was not reported previously. Above minus 4 degree C two phases, labeled N and M appear. The dividing line betwen these two phases is at approximately 18 wt. % DSCG and depends slightly on temperature. Phase N which prevails on the low concentration side of the dividing line seems to be smectic-like. There is no discontinuity in the ordering characteristics of the water solvent in the N-M transition region, suggesting that these two phases have similar ordered structures. These are believed to be columns of stacked highly hydrated DSCG molecules. In the M-phase these columns are arranged in ordered arrays, whereas in the N-phase they are relatively free to move within the bulk water while retaining their orientational order. The phase transition from the N,M phases to phase III is accompanied by a marked change in the water ordering indicating that the smectic structures in the latter phase are quite distinct from those in the N, M phases. Sample rotation experiments in a magnetic field show that all three phases are uniaxial and of type II (i. e. DELTA // chi less than 0) indicating that in all the lyotropic structures the DSCG molecules prefer an orientation in which the aromatic planes lie perpendicular to the director.