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Frequency-domain electron nuclear double resonance (ENDOR), two time-domain electron nuclear double resonance techniques, and electron spin echo envelope modulation spectroscopy are compared with respect to their merit in measurements of small hyperfine couplings to nuclei with intermediate gyromagnetic ratio such as 31P. The frequency-domain Mims ENDOR experiment is found to provide the most faithful line shapes. In the limit of long electron-nuclear distances of more than 0.5 nm, sensitivity of this experiment is optimized by matching the first interpulse delay to the transverse relaxation time of the electron spins. In the same limit, Mims ENDOR efficiency scales inversely with the sixth power of distance. Hyperfine splittings as small as 33 kHz can be detected, corresponding to an electron-31P distance of 1 nm. In systems, where a certain kind of nuclei is distributed in a plane, measurements of intermolecular hyperfine couplings can be analyzed in terms of a distance of closest approach of a paramagnetic center to that plane. By applying this technique to spin-labeled lipids in a fully hydrated lipid bilayer it is found that for a fraction of lipids, chain tilt angles can be 25° larger than the mean tilt angle of the lipid chains. This model of all-trans hydrocarbon chains with a broad distribution of tilt angles is also consistent with orientation selection effects in high-field ENDOR spectra. © 2005 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Concanavalin A is a member of the plant hemeagglutinin (or plant lectin) family that contains two metal binding sites; one, called S1, is occupied by Mn2+ and the other, S2, by Ca2+. 55Mn electron—nuclear double resonance (ENDOR) measurements were performed on a single crystal of concanavalin A at W-band (95 GHz, ~3.5 T) to determine the 55Mn nuclear quadrupole interaction in a protein binding site and its relation to structural parameters. Such measurements are easier at a high field because of the high sensitivity for size-limited samples and the reduction of second-order effects on the spectrum which simplifies spectral analysis. The analysis of the 55Mn ENDOR rotation patterns showed that two chemically inequivalent Mn2+ types are present at low temperatures, although the high-resolution X-ray structure reported only one site. Their quadrupole coupling constants, e2Qq/h, are significantly different; 10.7 ± 0.6 MHz for MnA2+ and only -2.7 ± 0.6 MHz for MnB2+. The ENDOR data also refined the hyperfine coupling determined earlier by single-crystal EPR measurements, yielding a small but significant difference between the two: -262.5 MHz for MnA2+ and -263.5 MHz for MnB2+. The principal z-axis for MnA2+ is not aligned with any of the Mn-ligand directions, but is 25° off the Mn-asp10 direction, and its orientation is different than that of the zero-field splitting (ZFS) interaction. Because of the small quadrupole interaction of MnB2+ the orientation dependence was very mild, leading to larger uncertainties in the asymmetry parameter. Nonetheless, there too z is not along the Mn-ligand bonds and is rotated 90° with respect to MnA. These results show, that similar to the ZFS, the quadrupolar interaction is highly sensitive to small differences in the coordination sphere of the Mn2+, and the resolution of the two types is in agreement with the earlier observation of a two-site conformational dynamic detected through the ZFS interaction, which is frozen out at low temperatures and averaged at room temperature. To account for the structural origin of the different e2Qq/h values, the electric field gradient tensor was calculated using the point-charge model. The calculations showed that a relatively small displacement of the oxygen ligand of asp10 can lead to differences on the order observed experimentally. [ABSTRACT FROM AUTHOR]

Mn 2+ often serves as a paramagnetic substitute to Mg 2+ , providing means for exploring the close environment of Mg 2+ 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 Mg 2+ -ATP to the ATPase active site. In this context, it is important to distinguish between the Mn 2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn 2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band 31 P 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 15 N- 31 P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn 2+ 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 55 Mn- 31 P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with 15 N- 31 P correlations. [ABSTRACT FROM AUTHOR]

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

Abstract: High resolution pulse EPR methods are usually applied to resolve weak magnetic electron-nuclear or electron–electron interactions that are otherwise unresolved in the EPR spectrum. Complete information regarding different magnetic interactions, namely, principal components and orientation of principal axis system with respect to the molecular frame, can be derived from orientation selective pulsed EPR measurements that are performed at different magnetic field positions within the inhomogeneously broadened EPR spectrum. These experiments are usually carried out consecutively, namely a particular field position is chosen, data are accumulated until the signal to noise ratio is satisfactory, and then the next field position is chosen and data are accumulated. Here we present a new approach for data acquisition of pulsed EPR experiments referred to as parallel acquisition. It is applicable when the spectral width is much broader than the excitation bandwidth of the applied pulse sequence and it is particularly useful for orientation selective pulse EPR experiments. In this approach several pulse EPR measurements are performed within the waiting (repetition) time between consecutive pulse sequences during which spin lattice relaxation takes place. This is achieved by rapidly changing the main magnetic field, B 0, to different values within the EPR spectrum, performing the same experiment on the otherwise idle spins. This scheme represents an efficient utilization of the spectrometer and provides the same spectral information in a shorter time. This approach is demonstrated on W-band orientation selective electron-nuclear double resonance (ENDOR), electron spin echo envelope modulation (ESEEM), electron–electron double resonance (ELDOR) – detected NMR and double electron–electron resonance (DEER) measurements on frozen solutions of nitroxides. We show that a factors of 3–6 reduction in total acquisition time can be obtained, depending on the experiment applied. [Copyright &y& Elsevier]

Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 Å, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and ¹H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the ¹H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the MnO bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites. [ABSTRACT FROM AUTHOR]

Summary We have applied high-field (W-band) pulse electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (EDNMR) to characterize the coordination sphere of the Mn 2+ co-factor in the nucleotide binding sites (NBSs) of ABC transporters. MsbA and BmrCD are two efflux transporters hypothesized to represent divergent catalytic mechanisms. Our results reveal distinct coordination of Mn 2+ to ATP and transporter residues in the consensus and degenerate NBSs of BmrCD. In contrast, the coordination of Mn 2+ at the two NBSs of MsbA is similar, which provides a mechanistic rationale for its higher rate constant of ATP hydrolysis relative to BmrCD. Direct detection of vanadate ion, trapped in a high-energy post-hydrolysis intermediate, further supports the notion of asymmetric hydrolysis by the two NBSs of BmrCD. The integrated spectroscopic approach presented here, which link energy input to conformational dynamics, can be applied to a variety of systems powered by ATP turnover. [ABSTRACT FROM AUTHOR]