Publications

Half-Integer High Spin (HIHS) systems with zero-field splitting (ZFS) parameters below 1 GHz are generally dominated by the spin |─1/2>→|+1/2 > central transition (CT). Accordingly, most pulsed Electron Paramagnetic Resonance (EPR) experiments are performed at this position for maximum sensitivity. However, in certain cases it can be desirable to detect higher spin transitions away from the CT in such systems. Here, we describe the use of frequency swept Wideband, Uniform Rate, Smooth Truncation (WURST) pulses for transferring spin population from the CT, and other transitions, of Gd(III) to the neighbouring higher spin transition |─3/2>→|─1/2 > at Q- and W-band frequencies. Specifically, we demonstrate this approach to enhance the sensitivity of ¹H Mims Electron-Nuclear Double Resonance (ENDOR) measurements on two model Gd(III) aryl substituted 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) complexes, focusing on transitions other than the CT. We show that an enhancement factor greater than 2 is obtained for both complexes at Q- and W-band frequencies by the application of two polarising pulses prior to the ENDOR sequence. This is in agreement with simulations of the spin dynamics of the system during WURST pulse excitation. The technique demonstrated here should allow more sensitive experiments to be measured away from the CT at higher operating temperatures, and be combined with any relevant pulse sequence.

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

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

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

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

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

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

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

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

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

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

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

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

Chirp and shaped pulses have been recently shown to be highly advantageous for improving sensitivity in DEER (double electron–electron resonance, also called PELDOR) measurements due to their large excitation bandwidth. The implementation of such pulses for pulse EPR has become feasible due to the availability of arbitrary waveform generators (AWG) with high sampling rates to support pulse shaping for pulses with tens of nanoseconds duration. Here we present a setup for obtaining chirp pulses on our home-built W-band (95 GHz) spectrometer and demonstrate its performance on Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements. We carried out an extensive optimization procedure on two model systems, Gd(III)-PyMTA–spacer–Gd(III)-PyMTA (Gd-PyMTA ruler; zero-field splitting parameter (ZFS) D ∼ 1150 MHz) as well as nitroxide–spacer–nitroxide (nitroxide ruler) to evaluate the applicability of shaped pulses to Gd(III) complexes and nitroxides, which are two important classes of spin labels used in modern DEER/EPR experiments. We applied our findings to ubiquitin, doubly labeled with Gd-DOTA-monoamide (D ∼ 550 MHz) as a model for a system with a small ZFS. Our experiments were focused on the questions (i) what are the best conditions for positioning of the detection frequency, (ii) which pump pulse parameters (bandwidth, positioning in the spectrum, length) yield the best signal-to-noise ratio (SNR) improvements when compared to classical DEER, and (iii) how do the sample's spectral parameters influence the experiment. For the nitroxide ruler, we report an improvement of up to 1.9 in total SNR, while for the Gd-PyMTA ruler the improvement was 3.1–3.4 and for Gd-DOTA-monoamide labeled ubiquitin it was a factor of 1.8. Whereas for the Gd-PyMTA ruler the two setups pump on maximum and observe on maximum gave about the same improvement, for Gd-DOTA-monoamide a significant difference was found. In general the choice of the best set of parameters depends on the D parameter of the Gd(III) complex.

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

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

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd-III spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-Gd-III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 mu M.

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.

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

DNP on heteronuclear spin systems often results in interesting phenomena such as the polarization enhancement of one nucleus during MW irradiation at the "forbidden" transition frequencies of another nucleus or the polarization transfer between the nuclei without MW irradiation. In this work we discuss the spin dynamics in a four-spin model system of the form {e_a-e_b-(¹H,¹³C)}, with the Larmor frequencies ω_a, ω_b, ω_H and ω_C, by performing Liouville space simulations. This spin system exhibits the common ¹H solid effect (SE), ¹³C cross effect (CE) and in addition high order CE-DNP enhancements. Here we show, in particular, the "proton shifted ¹³C-CE" mechanism that results in ¹³C polarization when the model system, at one of its ¹³C-CE conditions, is excited by a MW field at the zero quantum or double quantum electron-proton transitions ω_MW = ω_a ± ω_H and ω_MW = ω_b ± ω_H. Furthermore, we introduce the "heteronuclear" CE mechanism that becomes efficient when the system is at one of its combined CE conditions |ω_a - ω_b| = |ω_H ± ω_C|. At these conditions, simulations of the four-spin system show polarization transfer processes between the nuclei, during and without MW irradiation, resembling the polarization exchange effects often discussed in the literature. To link the "microscopic" four-spin simulations to the experimental results we use DNP lineshape simulations based on "macroscopic" rate equations describing the electron and nuclear polarization dynamics in large spin systems. This approach is applied based on electron-electron double resonance (ELDOR) measurements that show strong ¹H-SE features outside the EPR frequency range. Simulated ELDOR spectra combined with the indirect ¹³C-CE (iCE) mechanism, result in additional "proton shifted ¹³C-CE" features that are similar to the experimental ones. These features are also observed experimentally in ¹³C-DNP spectra of a sample containing 15 mM of trityl in a glass forming solution of ¹³C-glycerol/H₂O and are analyzed by calculating the basic ¹³C-SE and ¹³C-iCE shapes using simulated ELDOR spectra that were fitted to the experimental ones.

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

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.

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

The microwave frequency swept DNP enhancement, referred to as the DNP spectrum, is strongly dependent on the EPR spectrum of the polarizing radical and it reveals the underlying DNP mechanisms. Here we focus on two chlorinated trityl radicals that feature axially symmetric powder patterns at 95 GHz, the width of which are narrower than those of TEMPOL or TOTAPOL but broader than that of the trityl derivative OX63. The static DNP lineshapes of these commonly used radicals in DNP, have been recently analyzed in terms of a superposition of basic Solid Effect (SE) and Cross Effect (CE)-DNP lineshapes, with their relative contributions as a fit parameter. To substantiate the generality of this approach and further investigate an earlier suggestion that a ^35,37Cl-¹³C polarization transfer pathway, termed “hetero-nuclear assisted DNP”, 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 “hetero-nuclear assisted DNP” 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.

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

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

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

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

Modern pulse EPR experiments are routinely used to study the structural features of paramagnetic centers. They are usually performed at low temperatures, where relaxation times are long and polarization is high, to achieve a sufficient Signal/Noise Ratio (SNR). However, when working with samples whose amount and/or concentration are limited, sensitivity becomes an issue and therefore measurements may require a significant accumulation time, up to 12 h or more. As the detection scheme of practically all pulse EPR sequences is based on the integration of a spin echo - either primary, stimulated or refocused - a considerable increase in SNR can be obtained by replacing the single echo detection scheme by a train of echoes. All these echoes, generated by Carr-Purcell type sequences, are integrated and summed together to improve the SNR. This scheme is commonly used in NMR and here we demonstrate its applicability to a number of frequently used pulse EPR experiments: Echo-Detected EPR, Davies and Mims ENDOR (Electron-Nuclear Double Resonance), DEER (Electron-Electron Double Resonance|) and EDNMR (Electron-Electron Double Resonance (ELDOR)-Detected NMR), which were combined with a Carr-Purcell- Meiboom-Gill (CPMG) type detection scheme at W-band. By collecting the transient signal and integrating a number of refocused echoes, this detection scheme yielded a 1.6-5 folds SNR improvement, depending on the paramagnetic center and the pulse sequence applied. This improvement is achieved while keeping the experimental time constant and it does not introduce signal distortion.

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.

Proton Dynamic Nuclear Polarization (DNP) experiments were conducted on a 3.4 T homebuilt hybrid pulsed-EPR-NMR spectrometer, on static samples containing 10 mM or 40 mM TEMPOL in frozen glassy solutions of DMSO/water. During DNP experiments proton-NMR signals are enhanced with the help of microwave (MW) irradiation on or close to the Electron Paramagnetic Resonance (EPR) spectrum of the free radicals in the sample, transferring polarization from the free electrons to the nuclei. In the solid state a distinction is made between three DNP enhancement mechanisms: the Solid Effect (SE), the Cross Effect (CE) and Thermal Mixing (TM). In an effort to determine the dominant DNP mechanisms responsible for the enhancement of the nuclear signals, electron and nuclear spin-lattice relaxation rates, enhancement buildup times and microwave (MW) swept DNP spectra were measured as a function of temperature and MW irradiation strength. We observed lineshape variations of the DNP spectra that indicated changes in the relative contributions of SE-DNP and CE-DNP with temperature and MW power. Using a theoretical model describing the SE-DNP and CE-DNP the DNP spectra could be analyzed without involving the TM-DNP mechanism and the relative SE-DNP and CE-DNP contributions to the nuclear enhancement could be determined. From this analysis it follows that lowering the temperature beyond 20 K increases the SE-DNP and decreases the CE-DNP contributions. Possible explanations for this behavior are suggested.

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

The formation mechanism of the cubic mesoporous carbon, FDU-16, synthesized by evaporation-induced self-assembly (EISA) was investigated at the molecular level by electron paramagnetic resonance (EPR) spectroscopic techniques. This material is synthesized using F127 pluronic block copolymer [poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO ₁₀₆-PPO ₇₀-PEO ₁₀₆)] as a structure-directing agent (template) and phenolic resol as a carbon precursor. Using two spin probes derived from pluronics with PEO and PPO chains of different lengths that are designed to sense different regions of the system, we followed the evaporation and thermopolymerization stages of the synthesis in situ. To make such studies possible, we have used a polyurethane foam support, placed in the EPR tube, which allows for the efficient solvent evaporation as required for EISA. We focused on the evolution of the dynamics of the template and its interactions with the resol during the reaction. We observed that during the evaporation stage the resol is distributed throughout the entire PEO blocks, all the way to the PPO-PEO interface, interacting with them via H-bonds, thus hindering the local motion of the PEO chains. At the end of this stage there is no polarity gradient along the PEO blocks, as found for traditional F127 micelles in water or during the synthesis of silica materials, and the mesostructure is not well-defined. A polarity and a resol gradient developed during the thermopolymerization stage where the polymerizing resol is driven out to the outer region of the PEO corona. This produces a corona of resin-pluronic composite and a resol-free PPO core with high mobility of the PEO segments close to the PPO-PEO interface and restricted mobility in the composite corona. During this stage the final structure sets in.

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

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

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

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

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

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

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

We studied the structural evolution during the formation of large-pore cubic Ia3d silica-based mesoporous materials, synthesized with Pluroinc P123 and butanol as structure directing agents. We used cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM). Typically a silica precursor is added to an acid-catalyzed solution of Pluronic P123 and butanol. The latter serves as a cosolute, which can be added either at the beginning of the reaction, or after precipitation and the formation of a hexagonal phase. In this study we focused on the structural evolution from the hexagonal phase to the final cubic phase in the two different reactions. The same structural evolution with different kinetics was detected for both reactions. Cryo-HRSEM and FFR-TEM images revealed that from the hexagonal phase a perforated layer (PL) phase is formed, which later evolves into a bicontinuous structure. The final cubic phase forms within the layers, maintaining their orientation. We suggest a formation mechanism involving cylinder merging for the hexagonal to PL transition. Upon additional polymerization of the silica, the PL phase relaxes into the stable Ia3d cubic phase. Another minor mechanism detected involves the direct transition between the hexagonal to the final cubic phase through cylinder branching.

W-band (95 GHz) HYSCORE and pulse ENDOR are used to characterize the nitrosyl d₁ heme complex (d₁NO) of cd₁ nitrite reductase from Pseudomonas aeruginosa in the wild type and the Y10F mutant. The spectra and the derived ¹⁴N hyperfine and quadrupole interactions were found to be the same for wt and Y10F. This suggests that Tyr10 does not influence the NO ligand orientation in the reduced state in solution. This study is the first application of HYSCORE at high fields and shows its potential for characterizing low y nuclei with large hyperfine couplings.

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

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

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

This study focuses on the formation mechanism of the bicontinuous cubic Ia3̄d mesoporous material KIT-6, both on the molecular and on the mesoscopic levels. KIT-6 is synthesized with Pluronic P123 (PEO ₂₀PPO₇₀PEO₂₀), low acid concentration, and n-butanol at 40°C. Through in situ EPR measurements on a series of spin-labeled Pluronic molecules introduced at minute quantities into the reaction mixture, changes in the hydrophobicity and the mobility of the polymer chains during the reaction were observed. In addition, to learn more on the functionality of the butanol in this synthesis, freeze-quench electron spin-echo envelope modulation (ESEEM) measurements on reaction mixtures in D₂O and in butanol-d₁₀ were preformed. The above experiments gave information on variations in the butanol location and content in the micellar structures during the formation of KIT-6. The evolution of the solution nanostructures was determined by cryo-TEM. Five main stages were resolved: the first two occurred during the first 140 min of the reaction, where condensation of the silica oligomers takes place at the micellar/water interface; this induces depletion of water and butanol molecules from the core - corona interface and reduces the mobility of the ends of the Pluronic chains located at the corona - water interface. This in turn leads to a transition from spheroidal micelles to threadlike micelles and to their aggregation toward the end of the second stage. During the third stage, precipitation (140-160 min), reorganization in the micellar structure, and a change in the relative sizes of core and corona take place. The fourth stage, that ends around 6 h, involves the formation of a hexagonal phase, through accelerated condensation of silica oligomers in the corona, accompanied by extensive depletion of water and butanol molecules. The presence of butanol in the micelle corona is essential in the last stage, 6-24 h, where the cubic phase is formed. We show that the addition of butanol to the reaction mixture of SBA-15 after the formation of the hexagonal phase leads to the formation of the cubic phase.

The formation of templated mesoporous materials (TMM), where highly ordered mesoporous materials are prepared using surfactant self-assemblies as templates, is an intriguing process. It depends on a delicate interplay between several concomitant basic processes; the self-assembly of the surfactant molecules forming structures that serve as templates, the sol-gel chemistry that generates the inorganic silica network, and the specific interaction at the interface between the organic and forming inorganic phases. In this review we briefly describe the properties of TMM and review some basic principles underlying their formation mechanism. After a short description of the various methods that can be used to investigate the details of such reactions at the molecular level and the mesoscale we focus on the unique contribution of various EPR techniques. This is achieved by introducing nitroxide spin-probes, designed to examine different regions in the forming mesostructure, into the reaction mixture. Continuous wave (CW) EPR measurements, carried out in situ, give information on the polarity and microviscosity in the close environment of the spin-probe. These are complemented by electron-spin echo modulation (ESEEM) experiments that follow the water content, presence of additives and interaction with ions and provide an understanding of their effect on the structure of the final material. Finally, double electron-electron resonance (DEER) measurements are used to explore size variation of the micelles during the initial stages of the reaction.

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.

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

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

W-band (95 GHz) pulsed electron nuclear double resonance (ENDOR) measurements were carried out to determine quantitatively the first coordination shell of Mn²⁺ with ADP and ATPγS. The intensity of the ENDOR effect was used for counting the number of equivalent phosphate oxygens and water ligands. Titration curves for determining the binding constant of Mn ²⁺·ADP were obtained using the intensity of the X-band EPR spectrum and the ³¹P ENDOR effect. Both curves gave the same binding constant showing that phosphate ligand counting is plausible, provided that an appropriate reference is available. The comparison of the ³¹P ENDOR effect of the 1:1 ADP and ATPγS complexes shows that two phosphates are coordinated in both; while in ADP they are equivalent, in ATPγS they are slightly different. The reference system for water ligand counting was Mn(H ₂O)₆²⁺ in a H₂O-D₂O mixture. The results show a smaller error for the ²H ENDOR effect, compared to the ¹H ENDOR effect. Unlike the ³¹P ENDOR effect, the ¹H ENDOR effect dependence on [ADP] in the titration experiments showed that it is sensitive to variations in the zero-field splitting, which in turn alters the contributions of transitions other than the |-1/2〉 ↔ |1/2〉. This results in a larger error in the determination of the number of water ligands.

The properties of the Mn²⁺ site in the protein concanavalin A were investigated by single crystal W-band EPR/ENDOR (electron-nuclear double resonance) measurements. Initially, room temperature EPR measurements were carried out, one type of Mn²⁺ was identified and its zero-field splitting (ZFS) tensor was determined. In contrast, low temperature EPR measurements showed that two chemically inequivalent Mn²⁺ are present, Mn_A²⁺ and Mn_B²⁺, differing in their ZFS tensors. Variable temperature measurements revealed a two-site exchange between the two types. Although the dynamic process has been characterized by its rate and activation energy, just from the EPR measurements it was not possible to assign it to a specific residue. ¹H ENDOR measurements of the water and imidazole protons, which are the main contributors to the ENDOR spectra, showed only one type of signals, namely, they were not sensitive to the differences between Mn_A²⁺ and Mn _B²⁺. ⁵⁵Mn ENDOR spectra, which are dominated by the ⁵⁵Mn isotropic hyperfine, a_iso, and the nuclear quadrupole interaction did sense the differences. Analysis of the spectra recorded with the magnetic field along the crystallographic axes showed that the two have the same a_iso but different quadrupole tensors.

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

The properties of the silica layer during the formation of the mesoporous material MCM-41 were investigated by electron paramagnetic resonance (EPR) experiments carried out on a specifically designed, organo(trialkoxy)-silane spin probe, SL1SiEt. Minute amounts of the spin probe were co-condensed with the silica source, tetraethyl orthosilicate (TEOS), in the synthesis of MCM-41 with cetyltrimethylammonium bromide (CTAB) under basic conditions. The mobility and location of the spin probe were followed in the CTAB micellar solution before the reaction, in the reaction mixture and in the final ordered material. It was found that the EPR spectra of hydrolyzed SL1SiEt throughout the room temperature part of the reaction are characteristic of a fast tumbling species, indicating that the silica is highly fluid prior to drying. After filtering, a slow motion type spectrum was observed, showing that the spin-label experiences considerable motional hindrance. The liquidlike behavior could be restored upon stirring the material in water. When the reaction is performed with a hydrothermal stage, the spectrum of SL1SiEt in the final product is the same as that of the room temperature synthesized material, but the addition of water did not restore the high mobility, due to a higher degree of silica cross-linking. The location of SL1SiEt throughout the formation process was obtained from electron spin-echo envelope modulation (ESEEM) measurements on MCM-41 prepared with CTAB deuterated either at the N-methyl or the a position and in a reaction carried out in D₂O. Comparing the deuterium modulation depth, k(²H), induced by CTAB-α-d₂, CTAB-d₉, or D₂O in CTAB micellar solutions of a number of reference spin probes with those of SL1SiEt revealed that the hydrolyzed SL1SiEt is located near the polar heads of the surfactant in the absence of base and TEOS. This supports the postulation of charge matching at the interface as a driving force for the formation of the mesostructure. Similar experiments carried out on reaction mixtures containing SL1SiEt showed a decrease of k(²H) from CTAB-α-d₂ and CTAB-d₉ compared to the micellar solution, exhibiting practically no time dependence. This indicates that the spin probe is pulled away from the micelle-water interface into the loosely linked, forming silica network. After drying, the modulation depth induced by CTAB-α-d₂ and CTAB-d₉ increases, showing that, once the water is removed, the silica walls contract around the micelles, pushing the silica-linked spin probe into the organic phase within the mesopores.

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

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

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

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.

A combination of continuous wave (CW) electron paramagnetic resonance (EPR), pulsed EPR, and pulsed electron-nuclear double resonance (ENDOR) techniques were used to obtain structural information about the Cu2+ ions in hydrated, room-temperature evacuated, and dehydrated Cu-Y (Si:Al = 12 and 5) with a particular emphasis on framework Al interactions. W-band H-1 ENDOR was used to probe the water ligands, whereas X-band hyperfine sublevel correlation (HYSCORE) spectroscopic measurements were employed to detect Al-27 hyperfine couplings. The X-band CW EPR spectra show that a total of three Cu2+ species (A, B, and C) axe present in the samples. ENDOR measurements of hydrated and evacuated Cu-Y-12 indicate that species A and B have a complete coordination sphere of water and, on the basis of the absence of Al-27 signals in the HYSCORE spectrum, the Cu2+ is not directly bonded to the zeolite framework. Evacuation converted species B to species A. H-1 ENDOR spectra combined with simulations show that upon freezing, the equatorial and axial water ligands of species A and B have a distribution of orientations with respect to the Cu-O bond. The CW EPR spectrum of dehydrated Cu-Y-12 shows a single species (species C) and the HYSCORE spectrum exhibits cross-peaks from Al-27 with an isotropic coupling, a(iso), of 1.5 MHz. Unlike Cu-Y-12, evacuated Cu-Y-5 consists of species A and C, and the HYSCORE spectrum clearly shows a doublet of Al-27 cross-peaks with a(iso) = 3.0 MHz assigned to species C. Upon dehydration, the Al-27 coupling decreases to 2.6 MHz. This indicates that in species C the Cu2+ is bound to framework oxygens which are bonded to art Al nucleus. For Si:Al = 5, the zeolite framework becomes negative enough that it can replace water ligands even after mild evacuation. Simulations of Al-27 HYSCORE spectra indicate that species C is Cu2+ bound to the framework oxygens primarily near a single Al nucleus with a large quadrupole coupling constant.

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.

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).

Electron spin resonance, pulsed electron nuclear double resonance (ENDOR) spectroscopy at W- and X-band frequencies, and hyperfine sublevel correlation (HYSCORE) spectroscopy have been employed to determine the location of the V(IV) ions in H₄PVMo₁₁O₄₀ heteropolyacid catalysts. In these materials the heteropolyanions have the well-known structure of the Keggin molecule. Interactions of the unpaired electrons of the paramagnetic vanadyl ions (VO²⁺) with all relevant nuclei (^tH, ^3lP, and ⁵¹V) could be resolved. The complete analysis of the hyperfine coupling tensor for the phosphorus nucleus in the fourth coordination sphere of the V(IV) ion allowed for the first time a detailed structural analysis of the paramagnetic ions in heteropolyacids in hydrated and dehydrated catalysts. The ³¹P and ¹H ENDOR results show that V(IV) ions are incorporated as vanadyl pentaaqua complexes [VO(H₂O)₅]²⁺ in the void space between the heteropolyanions in the hydrated heteropolyacid. For the dehydrated H₄PVMo₁₁O₄₀ materials the distance between the V(IV) ion and the central phosphorus atom of the Keggin molecule could be determined with high accuracy on the basis of orientation-selective ³¹P ENDOR experiments and HYSCORE spectroscopy. The results give a first direct experimental evidence that the paramagnetic vanadium species are not incorporated at molybdenum sites into the Keggin structure of H₄PVMo₁₁O₄₀ and also do not act as bridges between two Keggin units after calcination of the catalyst. The vanadyl species are found to be directly attached to the Keggin molecules. The VO²⁺ ions are coordinated to four or three outer oxygen atoms from one PVMo₁₁O₄₀^-4 heteropolyanion in a trigonal-pyramidal or slightly distorted square-pyramidal coordination geometry, respectively.

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.

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

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

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

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

High frequency (95 GHz, W-band) pulsed ENDOR measurements were carried out on the ⁵⁷Fe-containing zeolites: Fe-sodalite (FeSOD), Fe-L (FeLTL), Fe-mazzite (FeMAZ), and Fe-ZSM5 (FeMFI), where ⁵⁷Fe(III) was introduced during synthesis. The echo-detected EPR spectra of all zeolites investigated, recorded at 1.8 K, show mainly the - ⁵/ ₂ > to - ³/ ₂ > EPR transition. Accordingly, the ENDOR spectra exhibit only two ⁵⁷Fe ENDOR transitions at 67.8-68.8 and 39.0-39.6 MHz, corresponding to M(S) = - ⁵/ ₂ and - ³/ ₂, respectively. From these frequencies isotropic hyperfine couplings of -29.0, -29.3, -29.5, and -29.6 MHz were derived for ⁵⁷FeSOD, ⁵⁷FeL, ⁵⁷FeMAZ, and ⁵⁷FeMFI, respectively. On the basis of an earlier assignment of the g = 2 signal in FeSOD to Fe(III) in tetrahedral framework sites it is concluded that hyperfine couplings in the range -29.0 to -29.6 MHz are characteristic of ⁵⁷Fe(III) in zeolite frameworks. In contrast to the X-band ⁵⁷Fe ENDOR signals, the W-band signals are free from second- and third-order contributions of the hyperfine and zero-field splitting (ZFS) interactions and are thus significantly simpler to assign and interpret. The ZFS contributions caused excessive inhomogeneous broadening of the X-band ENDOR spectra of ⁵⁷FeL, ⁵⁷FeMAz, and ⁵⁷FeMFI and the detection of the ENDOR spectra was practically impossible: All zeolites studied exhibited ENDOR signals from ²⁷Al and ⁵⁷FeSOD showed also clear ²³Na ENDOR signals. The hyperfine interaction of the ²³Na was significantly larger than that of the ²⁷Al, confirming the assignment of the Fe(III) to framework sites, substituting for Al. Moreover, the value obtained for the ²³Na anisotropic hyperfine component, 0.53 MHz, corresponding to a distance of 3.4 Å, is in a good agreement with the known structure of sodalite where the distance between a framework atom and the Na ⁺ cations in the center of the six rings is 3.35 Å. This work demonstrates the power and potential of high-field ENDOR in terms of resolution, signal assignment, and spectral analysis.

The Cu(II) sites in different preparations of tin oxide catalysts with low Cu(II) contents were characterized by EPR spectroscopy and electron spin echo envelope modulation (ESEEM) spectroscopy. The catalysts were prepared by two methods: (a) coprecipitation of a mixed oxide gel from aqueous solutions containing both tin(IV) and copper(II) ions and (b) by the sorption of Cu²⁺ cations onto tin(IV) oxide gel from aqueous solution. The samples were studied both before and after calcination. The EPR spectrum showed that from each type of preparation two major types of Cu(II) species, termed A and B, were generated. Prior to any thermal treatment the major species in both preparations was A, whereas after calcination at 573-1073 K the major species was B. Whilst the EPR spectrum of species A showed that it is static (on the EPR time scale) both at 100 K and at ambient temperatures, species B showed dynamic effects above 100 K which we attribute to a dynamic Jahn-Teller effect. The immediate environment of the Cu(II) was investigated in detail by following modulation from low-abundance ^117.119Sn nuclei and from ¹H nuclei in water and/or hydroxyl groups. In the latter we focused on the ¹H combination harmonics generated in the two- and four-pulse ESEEM experiments. From these experiments we concluded that in species A the Cu(II) is hydrated and situated on the external surface, coordinated either directly to a surface oxygen or via a hydrogen bond. In species B the Cu(II) is well incorporated into the SnO₂ lattice, it has very few protons in its vicinity, and some of the copper ions have an OH in their first coordination shell. This assignment was further substantiated by the inaccessibility of the Cu(II) species in B to adsorbed ammonia. The major difference between the two preparations is the significant amount of species B in the coprecipitated material prior to calcination.

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

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 “2+1” 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.

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

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

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.

Deuterium NMR spectra of deuterated probe molecules dissolved in several discotic liquid crystals possessing biaxial mesophases are reported. The spectra exhibit typical features characteristic of biaxial phases. This is particularly manifested through the asymmetry parameter of the average quadrupole tensor. The effect is demonstrated on two types of biaxial discotic phases: (i) a rectangular D_rd mesophase which appears in homologs of triphenylenehexa-n-alkanoates; and (ii) a tilted D_t mesophase which occurs in homologs of benzenehexa-n-alkanoates. The theory relating to the motional constants in biaxial phases and the asymmetry parameter of the quadrupole tensor (and other tensorial properties) is reviewed, and applied to some specific examples.

Deuterium NMR spectra of chain perdeuterated hexa-pentyloxy, -hexyloxy, -heptyloxy, and -octyloxy triphenylene (THE5, THE6, THE7, and THE8) were studied as functions of temperature in the mesophase region. The deuterium quadrupole splittings exhibited several characteristic features, in particular a steplike decrease in the splitting along the alkyl chain and an even-odd alteration in the methyl splittings within the homologous series. The results are interpreted in terms of possible conformational distributions of the alkyl chain. It is found that there is bending of the alkyl chains out of the aromatic plane, and a considerable degree of chain disorder.