Arnold M. Raitsimring

HYSCORE and DEER with an upgraded 95 GHz pulse EPR spectrometer

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 pi pulses of 25 ns and a microwave field, B(1)=0.71 mT. Such a high B(1) 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.

Selection of dipolar interaction by the “2 + 1” pulse train ESE

Abstract The new kind of electron spin echo (ESE), the “2 + 1” pulse train, is described. This method allows the measurement of dipole-dipole interactions between paramagnetic centers which are substantially weaker than those that can be measured by the ordinary two-pulse train. The dead time of ESE spectrometer response in this method is decreased to the duration of the first two pulses. The theory of dipole-dipole interaction in the ESE signal decay in the “2 + 1” pulse train is developed for different Flip rates and for different cases of spin spatial distribution. The theoretical data fit the experiment, carried out with model systems of H and D atoms, randomly distributed in the frozen solutions of sulfuric acid, and of biradicals. New data concerning the spatial distribution of radical clusters, resulting from y irradiation of the methanol, are given.

Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers

This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at K(a) band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59±12Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60Å, and analysis indicates that distance measurements up to 100Å are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

High-field pulsed EPR and ENDOR of Gd3+ complexes in glassy solutions

In this work D-band pulsed electron paramagnetic resonance was used to record the field-sweep spectra of several Gd3+ complexes in glassy water-methanol solutions. These spectra were analyzed by a specially developed stochastic superposition model that predicted the essential features of the distribution of the crystal field interaction (CFI) parameters in glassy systems. As a result of this analysis, the CFI distributions for the studied complexes were evaluated. The D-band Mims1H electron-nuclear double resonance spectra were free from CFI-related distortions, which allowed us to accurately determine the hyperfine interaction (HFI) parameters for water ligand protons and to unequivocally establish that the HFI distribution is solely related to the distribution of the Gd−H distances.

Electron spin echo envelope modulation theory for high electron spin systems in weak crystal field

Electron spin echo envelope modulation (ESEEM) experiments with aqueous complexes of Gd3+ and Mn2+ have shown that a common and unusual feature of the primary ESEEM spectra of such high spin/weak crystal field systems is an extremely low intensity of the sum combination line. Numerical simulations of the ESEEM spectra based on the existing theory [Coffino and Peisach, J. Chem. Phys. 97, 3072 (1992); Larsen and Singel, J. Chem. Phys. 98, 6704 (1993)] could not reproduce these ESEEM spectra. In this work the theoretical description of the ESEEM was revised and corrected, and new expressions were derived for the ESEEM from high electron spin (S>1/2) systems in a weak crystal field, interacting with a nuclear spin I=1/2. The corrections primarily affected the shape and intensity of the sum combination line, whose position was found to be sensitive to the product of the crystal field and anisotropic hyperfine interaction constants. These theoretical improvements resulted in a successful simulation of the prima...

NUCLEAR MODULATION EFFECTS IN 2+1 ELECTRON SPIN-ECHO CORRELATION SPECTROSCOPY

This work represents the synthesis of the 2+1 pulse sequence for the study of electron spin‐echo envelope modulation (ESEEM) with the technique of spin‐echo correlated spectroscopy (SECSY), which has previously been used to study nuclear modulation by two‐dimensional Fourier transform ESR methods. This example of ‘‘pulse adjustable’’ spectroscopy, wherein the pulse width and pulse amplitude of the second pulse in a three pulse sequence are introduced as adjustable parameters, leads to enhanced resolution to the key features of the nuclear modulation that are important for structural studies. This is demonstrated in studies on (i) a single crystal of irradiated malonic acid and (ii) a frozen solution of diphenylpicrylhydrazyl in toluene. In particular, it is shown for (i) how the nuclear modulation cross peaks can be preferentially enhanced relative to the autopeaks and to the matrix proton peaks, and also how the autopeaks can be significantly suppressed to enhance resolution for low‐frequency cross peaks...

The Mo−OH proton of the low-pH form of sulfite oxidase: Comparison of the hyperfine interactions obtained from pulsed ENDOR, CW-EPR and ESEEM measurements

Pulsed electron nuclear double resonance (ENDOR) spectra have been obtained for the exchangeable Mo-OH proton of the low-pH form of native chicken liver sulfite oxidase (SO) and recombinant human SO for the first time. The spectra of the two enzymes are very similar, indicating a similar binding geometry of the hydroxyl ligand to the Mo center. The isotropic hyperfine interaction (hfi) constant for the proton of the OH ligand in both enzymes is about 26 MHz. The anisotropic components of the hfi obtained from the pulsed ENDOR spectra are about 1.6–1.8 times larger than those obtained by continuous-wave electron paramagnetic resonance and electron spin echo envelope modulation. These hfi differences are explained by a rotational disorder of the Mo-OH group. A similar rotational disorder of the coordinated exchangeable ligand has been found previously for the high-pH and phosphate-inhibited forms of SO.

Zero field splitting fluctuations induced phase relaxation of Gd3+ in frozen solutions at cryogenic temperatures

Distance measurements using double electron-electron resonance (DEER) and Gd(3+) chelates for spin labels (GdSL) have been shown to be an attractive alternative to nitroxide spin labels at W-band (95GHz). The maximal distance that can be accessed by DEER measurements and the sensitivity of such measurements strongly depends on the phase relaxation of Gd(3+) chelates in frozen, glassy solutions. In this work, we explore the phase relaxation of Gd(3+)-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 |ms|. 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.

Investigation of the coordination structures of the molybdenum(v) sites of sulfite oxidizing enzymes by pulsed EPR spectroscopy

Sulfite oxidizing enzymes (SOEs) are physiologically vital and occur in all forms of life. During the catalytic cycle the five-coordinate square-pyramidal oxo-molybdenum active site passes through the Mo(v) state, and intimate details of the structure can be obtained from pulsed EPR spectroscopy through the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei (e.g., (1)H, (2)H, (17)O, (31)P) of the ligands. By employing spectrometer operational frequencies ranging from approximately 4 to approximately 32 GHz, it is possible to make the nuclear Zeeman interaction significantly greater than the hfi and nqi, and thereby simplify the interpretations of the spectra. The SOEs exhibit three general types of Mo(v) structures which differ in the number of nearby exchangeable protons (one, two or zero). The observed structure depends upon the organism, pH, anions in the medium, and method of reduction. One type of structure has a single exchangeable Mo-OH proton approximately in the equatorial plane and a large isotropic hfi (e.g., low pH form of chicken SOE, low pH form of plant SOE reduced by Ti(iii)); the second type has two exchangeable protons with distributed orientations out of the equatorial plane and very small (or zero) isotropic hfi (e.g., high pH form of chicken SOE, high pH form of plant SOE reduced by sulfite); the third type has no nearby exchangeable protons and a coordinated oxyanion (e.g., phosphate inhibited chicken SOE, low pH form of plant SOE reduced by sulfite). An additional structural conclusion is that the orientation angle of any exchangeable equatorial ligand (OH, OH(2), PO(4)(3-)) is not uniquely fixed, but is distributed around its central value by up to +/-20 degrees (depending on pH, the type of the ligand and the type of enzyme). An unexpected finding was that the axial oxo group of SOEs exchanges with (17)O in solutions enriched in H(2)(17)O. The first determination of oxo (17)O nqi parameters for a well-characterized model compound, [Mo(17)O(SPh)(4)](-), clearly demonstrated that (17)O nqi parameters can distinguish between oxo and OH(2) ligands.

Carr-Purcell train in the conditions of partial excitation of magnetic resonance spectrum

The present paper is stimulated by a technical realization of the CPT on a Bruker ESE spectrometer (6). The authors report here the results of calculations for the echo signals arising under partial excitation and treat the possibility of suppressing the unwanted (non-Carr-Purcell) echoes. The echo signal amplitudes may be readily calculated within the framework of the formalism proposed by Bloom (7)