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To characterise structure and order in the nanometer range, distances between electron spins and their distributions can be measured via dipolar spin-spin interactions by different pulsed electron paramagnetic resonance experiments. Here, for the single frequency technique for refocusing dipolar couplings (SIFTER), the buildup of dipolar modulation signal and intermolecular contributions is analysed for a uniform random distribution of monoradicals and biradicals in frozen glassy solvent by using the product operator formalism for electron spin S = 1/2. A dipolar oscillation artefact appearing at both ends of the SIFTER time trace is predicted, which originates from the weak coherence transfer between biradicals. The relative intensity of this artefact is predicted to be temperature independent, but to increase with the spin concentration in the sample. Different compositions of intermolecular backgrounds are predicted in the case of biradicals and in the case of monoradicals. We compare these predictions to experimental SIFTER traces for nitroxide and trityl monoradicals and biradicals. Our analysis demonstrates a good qualitative match with the proposed theoretical description. The resulting perspectives of quantitative analysis of SIFTER data are discussed. [ABSTRACT FROM AUTHOR]

Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the ¹H NMR spectrum of H2O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10-20 μM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum. [ABSTRACT FROM AUTHOR]

Our previous study on nitroxides in o-terphenyl (OTP) revealed two separable decoherence processes at low temperatures, best captured by the sum of two stretched exponentials (SSE) model. Dynamical decoupling (DD) extends both associated dephasing times linearly for 1 to 5 refocusing pulses [Soetbeer et al., Phys. Chem. Chem. Phys., 2018, 20, 1615]. Here we demonstrate an analogous DD behavior of water-soluble nitroxides in water–glycerol glass by using nitroxide and/or solvent deuteration for component assignment. Compared to the conventional Hahn experiment, we show that Carr–Purcell and Uhrig DD schemes are superior in resolving and identifying active dephasing mechanisms. Thereby, we observe a partial coherence loss to intramolecular nitroxide and trityl nuclei that can be alleviated, while the zero field splitting-induced losses for gadolinium labels cannot be refocused and contribute even at the central transition of this spin-7/2 system. Independent of the studied spin system, Uhrig DD leads to a characteristic convex dephasing envelope in both protonated water–glycerol and OTP glass, thus outperforming the Carr–Purcell scheme. [ABSTRACT FROM AUTHOR]

Identification and quantification of redox‐active centers at relevant conditions for catalysis is pivotal to understand reaction mechanisms and requires development of advanced operando methods. Herein, we demonstrate operando EPR spectroscopy as an important technique to quantify the oxidation state of representative CrPO4 and EuOCl catalysts during propane oxychlorination, an attractive route for propylene production. In particular, we show that the space‐time‐yield of C3H6 correlates with the amount of Cr2+ and Eu2+ ions generated over the catalysts during reaction. These results provide a powerful strategy to gather quantitative understanding of selective alkane oxidation, which could potentially be extrapolated to other functionalization approaches and operating conditions. [ABSTRACT FROM AUTHOR]

The distal hydrogen bond (H-bond) in dioxygenbinding proteins is crucial for the discrimination of O2 with respect to CO or NO. We report the preparation and characterization of a series of ZnII porphyrins, with one of three meso-phenyl rings bearing both an alkyl-tethered proximal imidazole ligand and a heterocyclic distal H-bond donor connected by a rigid acetylene spacer. Previously, we had validated the corresponding CoII complexes as synthetic model systems for dioxygen-binding heme proteins and demonstrated the structural requirements for proper distal H-bonding to CoII-bound dioxygen. Here, we systematically vary the H-bond donor ability of the distal heterocycles, as predicted based on pKa values. The H-bond in the dioxygen adducts of the CoII porphyrins was directly measured by Q-band Davies- ENDOR spectroscopy. It was shown that the strength of the hyperfine coupling between the dioxygen radical and the distal H-atom increases with enhanced acidity of the H-bond donor. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, for example, in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with an exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity and data interpretation, thus it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in-cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques providing information on side chain dynamics and water accessibility using nitroxide probes. An updated optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.