Coherence Transfer by Passage Pulses in Electron Paramagnetic Resonance Spectroscopy

Linear passage pulses provide a simple approach to ultra-wideband electron paramagnetic resonance (EPR) spectroscopy. We show by numerical simulations that the efficiency of inversion of polarization or coherence order on a single transition by idealized passage pulses is an exponential function of critical adiabaticity during passage, which allows for defining an effective flip angle for fast passage. This result is confirmed by experiments on E′ centers in Herasil glass. Deviations from the exponential law arise due to relaxation and a distribution of the adiabaticity parameter that comes from inhomogeneity of the irradiation field. Such inhomogeneity effects as well as edge effects in finite sweep bands cause a distribution of dynamic phase shifts, which can be partially refocused in echo experiments. In multilevel systems, passage of several transitions leads to generation of coherence on formally forbidden transitions that can also be described by the concept of an effective flip angle. On the one hand, such transfer to coherence on forbidden transitions is a significant magnetization loss mechanism for dipole–dipole coupled electron spin pairs at distances below about 2 nm. On the other hand, it can potentially be harnessed for electron spin echo envelope modulation (ESEEM) experiments, where matching of the irradiation field strength to the nuclear Zeeman frequency leads to efficient generation of nuclear coherence and efficient back transfer to electron coherence on allowed transitions at high adiabaticity

Double Electron−Electron Resonance Measured Between Gd³⁺ Ions and Nitroxide Radicals

Double electron−electron resonance has attracted growing attention as a technique to study structure and conformational changes of biomacromolecules. Here, a new combination of paramagnetic labels is experimentally tested, one being a commonly used nitroxide radical, and the other being a Gd³⁺ ion. The Gd³⁺−nitroxide spin pair can serve as a good substitute for the nitroxide−nitroxide pair of spin labels and potentially provides a link to other experimental approaches dealing with structural information

Gd(III)-PyMTA Label Is Suitable for In-Cell EPR

Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd­(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of <i>Xenopus laevis</i> oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP₁₀CP₁₀CP₁₀-NH₂ was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP₁₀C­(Gd-PyMTA)­P₁₀C­(Gd-PyMTA)­P₁₀-NH₂, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into <i>Xenopus laevis</i> oocytes, and the Gd­(III)–Gd­(III) distances were determined by double electron–electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd­(III) ion and the C_α of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP₁₀C­(Gd-PyMTA)­P₁₀C­(Gd-PyMTA)­P₁₀-NH₂ is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix

Laser-Induced Magnetic Dipole Spectroscopy

Pulse electron paramagnetic resonance measurements of nanometer scale distance distributions have proven highly effective in structural studies. They exploit the magnetic dipole–dipole coupling between spin labels site-specifically attached to macromolecules. The most commonly applied technique is double electron–electron resonance (DEER, also called pulsed electron double resonance (PELDOR)). Here we present the new technique of laser-induced magnetic dipole (LaserIMD) spectroscopy based on optical switching of the dipole–dipole coupling. In a proof of concept experiment on a model peptide, we find, already at a low quantum yield of triplet excitation, the same sensitivity for measuring the distance between a porphyrin and a nitroxide label as in a DEER measurement between two nitroxide labels. On the heme protein cytochrome C, we demonstrate that LaserIMD allows for distance measurements between a heme prosthetic group and a nitroxide label, although the heme triplet state is not directly observable by an electron spin echo

High-Field Electron Paramagnetic Resonance and Density Functional Theory Study of Stable Organic Radicals in Lignin: Influence of the Extraction Process, Botanical Origin, and Protonation Reactions on the Radical <b>g</b> Tensor

The radical concentrations and <i>g</i> factors of stable organic radicals in different lignin preparations were determined by X-band EPR at 9 GHz. We observed that the <i>g</i> factors of these radicals are largely determined by the extraction process and not by the botanical origin of the lignin. The parameter mostly influencing the <i>g</i> factor is the pH value during lignin extraction. This effect was studied in depth using high-field EPR spectroscopy at 263 GHz. We were able to determine the <i>g</i>_<i>xx</i>, <i>g</i>_<i>yy</i>, and <i>g</i>_<i>zz</i> components of the <b>g</b> tensor of the stable organic radicals in lignin. With the enhanced resolution of high-field EPR, distinct radical species could be found in this complex polymer. The radical species are assigned to substituted <i>o</i>-semiquinone radicals and can exist in different protonation states <b>SH3+</b>, <b>SH2</b>, <b>SH1-</b>, and <b>S2-</b>. The proposed model structures are supported by DFT calculations. The <i>g</i> principal values of the proposed structure were all in reasonable agreement with the experiments

Changes in the Microenvironment of Nitroxide Radicals around the Glass Transition Temperature

For structural characterization by pulsed EPR methods, spin-labeled macromolecules are routinely studied at cryogenic temperatures. The equilibration of the conformational ensemble during shock-freezing occurs to a good approximation at the glass transition temperature (<i>T</i>_g). In this work, we used X-band power saturation continuous wave (cw) EPR to obtain information on the glass transition temperatures in the microenvironment of nitroxide radicals in solvents or bound to different sites in proteins. The temperature dependence of the saturation curve of nitroxide probes in pure glycerol or <i>ortho</i>-terphenyl showed detectable transitions at the respective <i>T</i>_g values, with the latter solvent characterized by a sharper change of the saturation properties, according to its higher fragility. In contrast, nitroxide probes in a glycerol/water mixture showed a discontinuity in the saturation properties close to the expected glass transition temperature, which made the determination of <i>T</i>_g complicated. Low-temperature W-band cw EPR and W-band ELDOR-detected NMR experiments demonstrated that the discontinuity is due to local rearrangements of H-bonds between water molecules and the nitroxide reporter group. The change in the network of H-bonds formed between the nitroxide and water molecules that occurs around <i>T</i>_g was found to be site-dependent in spin-labeled proteins. This effect can therefore be modulated by neighboring residues with different steric hindrances and/or charge distributions and possibly by the glycerol enrichment on protein surfaces. In conclusion, if the thermal history of the sample is carefully reproduced, the nitroxide probe is extremely sensitive in reporting site-specific changes in the H-bonding to water molecules close to <i>T</i>_g and local glass transition temperatures in spin-labeled macromolecules

Site-Specific Information on Membrane Protein Folding by Electron Spin Echo Envelope Modulation Spectroscopy

Compared to folding of soluble proteins, folding of membrane proteins is complicated by the fact that it requires an amphiphilic environment. Few existing techniques can provide structurally resolved information on folding kinetics. For the major plant light harvesting complex LHCII, it is demonstrated that changes in water accessibility of a particular amino acid residue can be followed during folding by measuring the hyperfine interaction of spin labels with deuterium nuclei of heavy water. The incorporation of residue 196 into the hydrophobic core of a detergent micelle was investigated. The technique provides a time constant that is similar to the one found with fluorescence spectroscopy for the slower folding step of the whole protein and with electron paramagnetic resonance for change of the distance between residues 90 and 196. If applied to several residues, this technique should provide information on the sequence of events during membrane protein folding

Multiple Pathway Relaxation Enhancement in the System Composed of Three Paramagnetic Species: Nitroxide Radical–Ln³⁺–O₂

Longitudinal relaxation of nitroxide spin-labels has been measured for a membrane-incorporated α-helical polypeptide in the presence and absence of residual amounts of membrane-dissolved O₂ and paramagnetic Dy³⁺ ions. Such a model system, containing three different types of paramagnetic species, provides an important example of nonadditivity of two different relaxation channels for the nitroxide spins

Electron Spin Density Distribution in the Special Pair Triplet of <i>Rhodobacter sphaeroides</i> R26 Revealed by Magnetic Field Dependence of the Solid-State Photo-CIDNP Effect

Photo-CIDNP (photochemically induced dynamic nuclear polarization) can be observed in frozen and quinone-blocked photosynthetic reaction centers (RCs) as modification of magic-angle spinning (MAS) NMR signal intensity under illumination. Studying the carotenoidless mutant strain R26 of <i>Rhodobacter sphaeroides</i>, we demonstrate by experiment and theory that contributions to the nuclear spin polarization from the three-spin mixing and differential decay mechanism can be separated from polarization generated by the radical pair mechanism, which is partially maintained due to differential relaxation (DR) in the singlet and triplet branch. At a magnetic field of 1.4 T, the latter contribution leads to dramatic signal enhancement of about 80 000 and dominates over the two other mechanisms. The DR mechanism encodes information on the spin density distribution in the donor triplet state. Relative peak intensities in the photo-CIDNP spectra provide a critical test for triplet spin densities computed for different model chemistries and conformations. The unpaired electrons are distributed almost evenly over the two moieties of the special pair of bacteriochlorophylls, with only slight excess in the L branch

Single Crystal Electron Paramagnetic Resonance of Dimethylammonium and Ammonium Hybrid Formate Frameworks: Influence of External Electric Field

We present a continuous wave electron paramagnetic resonance (EPR) study of a Mn²⁺ doped [(CH₃)₂NH₂]­[Zn­(HCOO)₃] hybrid dense metal–organic framework (MOF) that exhibits an order–disorder structural phase transition at <i>T</i>_c = 163 K. The W-band EPR measurements of a powder sample are performed to verify the previously reported spin Hamiltonian parameters of the Mn²⁺ centers in the low-temperature phase. The temperature dependent single crystal X-band EPR experiments reveal that Mn²⁺ probe ions are susceptible to the phase transition, as the spectrum changes drastically at <i>T</i>_c. The angular dependent EPR spectra of Mn²⁺ centers are obtained by rotating the single crystal sample about three distinct directions. The simulation of the determined angular dependences reveals six MnO₆ octahedra in the ordered phase that originate from a severe crystal twinning of the [(CH₃)₂NH₂]­[Zn­(HCOO)₃] MOF. The possible ferroelectric origin of the crystalline twins is investigated by single crystal EPR measurements with an applied external electric field. No significant effect of the electric field on the spectra is observed. The EPR results are supported by the measurements of the electric field dependence of the macroscopic electric polarization. Analogous EPR measurements are performed on a single crystal sample of ferroelectric Mn²⁺ doped [NH₄]­[Zn­(HCOO)₃] MOF. Contrary to the dimethylammonium framework, the EPR signal and electric polarization of the ammonium compound demonstrate clear ferroelectric behavior