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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]

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]

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]

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 studies on diamagnetic proteins are based on site-directed spin labelling and require relatively much effort for engineering cystein point mutations as well as expressing and labelling the protein. Therefore, it is advantageous to predict those sites and pairs of sites that can provide the most precise and most reliable information on accessibility and distances. Systematic site scans based on a rotamer library approach for spin label side groups allow for such predictions. Figures of merit are defined that can be used to rank sites according to their potential suitability in studies of structure and structural changes. Such site scans can still provide useful results if only a backbone model and the amino acid sequence of the protein are known. [ABSTRACT FROM AUTHOR]

Innentitelbild: Quantification of Redox Sites during Catalytic Propane Oxychlorination by Operando EPR Spectroscopy (Angew. Keywords: alkane activation; heterogeneous catalysis; operando EPR spectroscopy; propane oxychlorination; reaction mechanism EN alkane activation heterogeneous catalysis operando EPR spectroscopy propane oxychlorination reaction mechanism 3354 3354 1 02/10/21 20210215 NES 210215 B Operando-EPR wird heiß b Im Forschungsartikel auf S. 3640 demonstrieren Guido Zichittella, Yevhen Polyhach, Javier Pérez-Ramírez et al. die Operando-EPR-Spektroskopie als zentrale Technik zur Bestimmung des Oxidationszustands von CrPO SB 4 sb - und EuOCl-Katalysatoren während der katalytischen Hochtemperatur-Oxychlorierung von Propan. Alkane activation, heterogeneous catalysis, operando EPR spectroscopy, propane oxychlorination, reaction mechanism. [Extracted from the article]

To characterize structure and molecular order in the nanometre 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 the intermolecular background are predicted in the case of biradicals and in the case of monoradicals. Our theoretical account suggests that the appropriate procedure of extracting the intramolecular dipolar contribution (form factor) requires fitting and subtracting the unmodulated part, followed by division by an intermolecular background function that is different in shape. This scheme differs from the previously used heuristic background division approach. We compare our theoretical derivations 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 for a quantitative analysis of SIFTER data are discussed. [ABSTRACT FROM AUTHOR]

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]

The available structural information on LeuT and structurally related transporters suggests that external loop 4 (eL4) and the outer end of transmembrane domain (TM) 10' participate in the reversible occlusion of the outer pathway to the solute binding sites. Here, the functional significance of eL4 and the outer region of TM10' are explored using the sodium/proline symporter PutP as a model. Glu-311 at the tip of eL4, and various amino acids around the outer end of TM10' are identified as particularly crucial for function. Substitutions at these sites inhibit the transport cycle, and affect in part ligand binding. In addition, changes at selected sites induce a global structural alteration in the direction of an outward-open conformation. It is suggested that interactions between the tip of eL4 and the peptide backbone at the end of TM10' participate in coordinating conformational alterations underlying the alternating access mechanism of transport. Together with the structural information on LeuT-like transporters, the results further specify the idea that common design and functional principles are maintained across different transport families. [ABSTRACT FROM AUTHOR]

The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photosynthesis, and in preventing photodamage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment are only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is introduced to prepare LHCII trimers in which only one monomer is spin-labeled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron spin echo envelope modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the "Velcro" hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kühlbrandt, W. (2005)EMBOJ. 24, 919'928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound. [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]

Nucleotidyl cyclases, includingmembrane-integral and soluble adenylyl and guanylyl cyclases, are central components in a wide range of signaling pathways. These proteins are architecturally diverse, yet many of them share a conserved feature, a helical region that precedes the catalytic cyclase domain. The role of this region in cyclase dimerization has been a subject of debate. Although mutations within this region in various cyclases have been linked to genetic diseases, the molecular details of their effects on the enzymes remain unknown. Here, we report an X-ray structure of the cytosolic portion of the membrane-integral adenylyl cyclase Cya from Mycobacterium intracellulare in a nucleotide-bound state. The helical domains of each Cya monomer form a tight hairpin, bringing the two catalytic domains into an active dimerized state. Mutations in the helical domain of Cya mimic the disease-related mutations in human proteins, recapitulating the profiles of the corresponding mutated enzymes, adenylyl cyclase-5 and retinal guanylyl cyclase-1. Our experiments with fulllength Cya and its cytosolic domain link the mutations to protein stability, and the ability to induce an active dimeric conformation of the catalytic domains. Sequence conservation indicates that this domain is an integral part of cyclase machinery across protein families and species. Our study provides evidence for a role of the helical domain in establishing a catalytically competent dimeric cyclase conformation. Our results also suggest that the disease-associated mutations in the corresponding regions of human nucleotidyl cyclases disrupt the normal helical domain structure. [ABSTRACT FROM AUTHOR]

Summary: The Na+/proline symporter (PutP), like several other Na+-coupled symporters, belongs to the so-called LeuT-fold structural family, which features ten core transmembrane domains (cTMs) connected by extra- and intracellular loops. The role of these loops has been discussed in context with the gating function in the alternating access model of secondary active transport processes. Here we report the complete spin-labeling site scan of extracellular loop 4 (eL4) in PutP that reveals the presence of two α-helical segments, eL4a and eL4b. Among the eL4 residues that are directly implicated in the functional dynamics of the transporter, Phe314 in eL4b anchors the loop by means of hydrophobic contacts to cTM1 close to the ligand binding sites. We propose that ligand-induced conformational changes at the binding sites are transmitted via the anchoring residue to eL4 and through eL4 further to adjacent cTMs, leading to closure of the extracellular gate. [ABSTRACT FROM AUTHOR]

Abstract: Double electron electron resonance (DEER) spectroscopy has been established as a valuable method to determine distances between spin labels bound to protein molecules. Caused by selective excitation of molecular orientations DEER primary data also depend on the mutual orientation of the spin labels. For a doubly spin labeled variant of the cytoskeletal protein vinculin tail strong orientation selection can be observed already at X-band frequencies, which allows us to reduce the problem to the relative orientation of two molecular axes and the spin–spin axis parameterized by three angles. A full grid search of parameter space reveals that the DEER experiment introduces parameter-space symmetry higher than the symmetry of the spin Hamiltonian. Thus, the number of equivalent parameter sets is twice as large as expected and the relative orientation of the two spin labels is ambiguous. Except for this inherent ambiguity the most probable relative orientation of the two spin labels can be determined with good confidence and moderate uncertainty by global fitting of a set of five DEER experiments at different offsets between pump and observer frequency. The experiment provides restraints on the angles between the z axis of the nitroxide molecular frame and the spin–spin vector and on the dihedral between the two z axes. When using the same type of label at both sites, assignment of the angle restraints is ambiguous and the sign of the dihedral restraint is also ambiguous. [Copyright &y& Elsevier]

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations. [ABSTRACT FROM AUTHOR]

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfate- solubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly. [ABSTRACT FROM AUTHOR]