Ilia Kaminker

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.

Spectroscopic selection of distance measurements in a protein dimer with mixed nitroxide and Gd3+ spin labels

The pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd(3+) tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd(3+) ion, and two Gd(3+) ions. Gd(3+)-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

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

W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity.

Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd(3+) chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd(3+) central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 μs in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd(3+)-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.

A Dynamic Nuclear Polarization spectrometer at 95 GHz/144 MHz with EPR and NMR excitation and detection capabilities

A spectrometer specifically designed for systematic studies of the spin dynamics underlying Dynamic Nuclear Polarization (DNP) in solids at low temperatures is described. The spectrometer functions as a fully operational NMR spectrometer (144 MHz) and pulse EPR spectrometer (95 GHz) with a microwave (MW) power of up to 300 mW at the sample position, generating a MW B(1) field as high as 800 KHz. The combined NMR/EPR probe comprises of an open-structure horn-reflector configuration that functions as a low Q EPR cavity and an RF coil that can accommodate a 30-50 μl sample tube. The performance of the spectrometer is demonstrated through some basic pulsed EPR experiments, such as echo-detected EPR, saturation recovery and nutation measurements, that enable quantification of the actual intensity of MW irradiation at the position of the sample. In addition, DNP enhanced NMR signals of samples containing TEMPO and trityl are followed as a function of the MW frequency. Buildup curves of the nuclear polarization are recorded as a function of the microwave irradiation time period at different temperatures and for different MW powers.

High‐Field Pulsed EPR Spectroscopy for the Speciation of the Reduced [PV2Mo10O40]6− Polyoxometalate Catalyst Used in Electron‐Transfer Oxidations

An in-depth spectroscopic EPR investigation of a key intermediate, formally notated as [PV(IV)V(V)Mo(10)O(40)](6-) and formed in known electron-transfer and electron-transfer/oxygen-transfer reactions catalyzed by H(5)PV(2)Mo(10)O(40), has been carried out. Pulsed EPR spectroscopy have been utilized: specifically, W-band electron-electron double resonance (ELDOR)-detected NMR and two-dimensional (2D) hyperfine sub-level correlation (HYSCORE) measurements, which resolved (95)Mo and (17)O hyperfine interactions, and electron-nuclear double resonance (ENDOR), which gave the weak (51)V and (31)P interactions. In this way, two paramagnetic species related to [PV(IV)V(V)Mo(10)O(40)](6-) were identified. The first species (30-35 %) has a vanadyl (VO(2+))-like EPR spectrum and is not situated within the polyoxometalate cluster. Here the VO(2+) was suggested to be supported on the Keggin cluster and can be represented as an ion pair, [PV(V)Mo(10)O(39)](8-)[V(IV)O(2+)]. This species originates from the parent H(5)PV(2)Mo(10)O(40) in which the vanadium atoms are nearest neighbors and it is suggested that this isomer is more likely to be reactive in electron-transfer/oxygen-transfer reaction oxidation reactions. In the second (70-65 %) species, the V(IV) remains embedded within the polyoxometalate framework and originates from reduction of distal H(5)PV(2)Mo(10)O(40) isomers to yield an intact cluster, [PV(IV)V(V)Mo(10)O(40)](6-).

Increasing sensitivity of pulse EPR experiments using echo train detection schemes.

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 12h 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.

Determination of the 14N quadrupole coupling constant of nitroxide spin probes by W-band ELDOR-detected NMR

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

Simultaneous acquisition of pulse EPR orientation selective spectra.

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.

Oxidation of carbon monoxide cocatalyzed by palladium(0) and the H(5)PV(2)Mo(10)O(40) polyoxometalate probed by electron paramagnetic resonance and aerobic catalysis.

The H(5)PV(2)Mo(10)O(40) polyoxometalate and Pd/Al(2)O(3) were used as co-catalysts under anaerobic conditions for the activation and oxidation of CO to CO(2) by an electron transfer-oxygen transfer mechanism. Upon anaerobic reduction of H(5)PV(2)Mo(10)O(40) with CO in the presence of Pd(0) two paramagnetic species were observed and characterized by continuous wave electron paramagnetic resonance (CW-EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopic measurements. Major species I (65-70%) is assigned to a species resembling a vanadyl cation that is supported on the polyoxometalate and showed a bonding interaction with (13)CO. Minor species II (30-35%) is attributed to a reduced species where the vanadium(IV) atom is incorporated in the polyoxometalate framework but slightly distanced from the phosphate core. Under aerobic conditions, CO/O(2), a nucleophilic oxidant was formed as elucidated by oxidation of thianthrene oxide as a probe substrate. Oxidation reactions performed on terminal alkenes such as 1-octene yielded a complicated mixture of products that was, however, clearly a result of alkene epoxidation followed by subsequent reactions of the intermediate epoxide. The significant competing reaction was a hydrocarbonylation reaction that yielded a approximately 1:1 mixture of linear/branched carboxylic acids.