Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance.

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

Temperature-cycle electron paramagnetic resonance.

We report on a novel approach to the study of rates and short-lived intermediates of (bio)chemical reactions that involve paramagnetic species. Temperature-cycle Electron Paramagnetic Resonance (EPR) concerns the repeated heating of a reaction mixture in the cavity of an EPR spectrometer by pulsed irradiation with a near-infrared diode laser combined with intermittent characterization of the sample by 275 GHz EPR at a lower temperature at which the reaction does not proceed. The new technique is demonstrated for the reduction of TEMPOL with sodium dithionite in aqueous solution down to the sub-second time scale. We show that a single sample suffices to obtain a complete kinetic trace. Variation of the length and power of the laser pulse offers great flexibility as regards the time scale of the experiment and the temperature at which the reaction can be studied. For water/glycerol mixtures we introduce a simple way to obtain and load an unreacted sample into the spectrometer at low temperature.

Correction: Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy.

Correction for 'Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy' by Janne Soetbeer et al., Phys. Chem. Chem. Phys., 2018, 20, 25506-25517.

Analysis of the EPR spectra of transferrin: the importance of a zero-field-splitting distribution and 4th-order terms.

Multi-frequency EPR spectroscopy can provide high-level structural information on high-spin Fe3+ sites in proteins and enzymes. Unfortunately, analysis of the EPR spectra of these spin systems is hindered by the presence of broad distributions in the zero-field-splitting (ZFS) parameters, which reflect conformational heterogeneity of the iron sites. We present the analysis of EPR spectra of high-spin Fe3+ bound to human serum transferrin. We apply a method termed the grid-of-errors to extract the distributions of the individual ZFS parameters from EPR spectra recorded in the high-field limit at a microwave frequency of 275 GHz. Study of a series of transferrin variants shows that the ZFS distributions are as characteristic of the structure of a high-spin Fe3+ site as the ZFS parameters themselves. Simulations based on the extracted ZFS distributions reproduce spectra recorded at 34 GHz (Q band) and 9.7 GHz (X band), including subtle variations that were previously difficult to quantify. The X-band spectrum of transferrin shows a characteristic double peak, which has puzzled researchers for decades. We show that the double peak is uniquely related to the term B4-3O4-3(S) in the spin Hamiltonian. Our method is generally applicable in the analysis of spectra that arise from a broad distribution of parameters.

Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy.

The chemical structure of polarizing agents critically determines the efficiency of dynamic nuclear polarization (DNP). For cross-effect DNP, biradicals are the polarizing agents of choice and the interaction and relative orientation of the two unpaired electrons should be optimal. Both parameters are affected by the molecular structure of the biradical in the frozen glassy matrix that is typically used for DNP/MAS NMR and likely differs from the structure observed with X-ray crystallography. We have determined the conformations of six bis-nitroxide polarizing agents, including the highly efficient AMUPol, in their DNP matrix with EPR spectroscopy at 9.7 GHz, 140 GHz, and 275 GHz. The multi-frequency approach in combination with an advanced fitting routine allows us to reliably extract the interaction and relative orientation of the nitroxide moieties. We compare the structures of six bis-nitroxides to their DNP performance at 500 MHz/330 GHz.

Sensing the framework state and guest molecules in MIL-53(Al) via the electron paramagnetic resonance spectrum of VIV dopant ions.

X-ray diffraction (XRD) and electron paramagnetic resonance spectroscopy (EPR) were combined to study the structural transformations induced by temperature, pressure and air humidity of the "breathing" metal-organic framework (MOF) MIL-53(Al), doped with paramagnetic VIV ions, after activation. The correlation between in situ XRD and thermogravimetric analysis measurements showed that upon heating this MOF in air, starting from ambient temperature and pressure, the narrow pore framework first dehydrates and after that makes the transition to a large pore state (lp). The EPR spectra of VIV[double bond, length as m-dash]O molecular ions, replacing Al-OH in the structure, also allow to distinguish the as synthesized, hydrated (np-h) and dehydrated narrow pore (np-d), and lp states of MIL-53(Al). A careful analysis of EPR spectra recorded at microwave frequencies between 9.5 and 275 GHz demonstrates that all VIV[double bond, length as m-dash]O in the np-d and lp states are equivalent, whereas in the np-h state (at least two) slightly different VIV[double bond, length as m-dash]O sites exist. Moreover, the lp MIL-53(Al) framework is accessible to oxygen, leading to a notable broadening of the VIV[double bond, length as m-dash]O EPR spectrum at pressures of a few mbar, while such effect is absent for the np-h and np-d states for pressures up to 1 bar.

Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles.

Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.

The magnetic field dependence of cross-effect dynamic nuclear polarization under magic angle spinning.

We develop a theoretical description of Dynamic Nuclear Polarization (DNP) in solids under Magic Angle Spinning (MAS) to describe the magnetic field dependence of the DNP effect. The treatment is based on an efficient scheme for numerical solution of the Liouville-von Neumann equation, which explicitly takes into account the variation of magnetic interactions during the sample spinning. The dependence of the cross-effect MAS-DNP on various parameters, such as the hyperfine interaction, electron-electron dipolar interaction, microwave field strength, and electron spin relaxation rates, is analyzed. Electron spin relaxation rates are determined by electron paramagnetic resonance measurements, and calculations are compared to experimental data. Our results suggest that the observed nuclear magnetic resonance signal enhancements provided by MAS-DNP can be explained by discriminating between "bulk" and "core" nuclei and by taking into account the slow DNP build-up rate for the bulk nuclei.

Exploring the Fe(III) binding sites of human serum transferrin with EPR at 275 GHz.

We report 275 GHz EPR spectra of human serum transferrin. At this high microwave frequency the zero-field splitting between the magnetic sublevels of the high-spin [Formula: see text] sites can be accurately determined. We find the zero-field splitting to be a sensitive probe of the structure of the transferrin iron-binding sites. Signals arising from iron bound to the transferrin N-lobe can clearly be distinguished from signals from iron bound to the C-lobe. Moreover, our spectra show that the structure of the iron site in the N-lobe is influenced by the presence and conformation of the C-lobe. The spectra of a series of N-lobe mutants altering the second-shell interaction of Arg124 with the synergistic anion carbonate reflect conformational changes induced at the iron site.

The type 1 copper site of pseudoazurin: axial and rhombic.

We report on a high-frequency electron-paramagnetic-resonance study of the type 1 copper site of pseudoazurin. The spectra fully resolve the contribution of a nearly axial spectrum besides the rhombic spectrum, which unequivocally proves the existence of two conformations of the copper site. Pseudoazurins have been considered from Achromobacter cycloclastes including eight mutants and from Alcaligenes faecalis. The two conformations are virtually the same for all pseudoazurins, but the rhombic/axial population varies largely, between 91/9 and 33/67. These observations are discussed in relation to optical absorption spectra and X-ray diffraction structures. A similar observation for fern plastocyanin from Dryopteris crassirhizoma suggests that dual conformations of type 1 copper sites are more common.

Multifrequency EPR study of Fe3+ and Co2+ in the active site of desulforedoxin.

The understanding of the electronic structure of S > 1/2 transition-metal sites that show a large zero-field splitting (ZFS) of the magnetic sublevels benefits greatly from study by electron-paramagnetic-resonance (EPR) spectroscopy at frequencies above the standard 9.5 GHz. However, high-frequency EPR spectroscopy is technically challenging and still developing. Particularly the sensitivity of high-frequency EPR spectrometers is often too low to apply the technique in the study of transition-metal sites in proteins and enzymes. Here we report a multifrequency EPR study (at 9.5, 94.9, and 275.7 GHz) of the active site of the protein desulforedoxin, both in its natural Fe(3+) form and substituted with Co(2+). The 275.7 GHz EPR spectra made it possible to determine the ZFS parameters of the Fe(3+) site with high precision. No 275.7 GHz spectrum could be observed of the Co(2+) site, but based on 9.5 GHz spectra, its ZFS parameters could be estimated. We find that the typical variation in the geometry of the active site of a protein or enzyme, referred to as conformational strain, does not only make the detection of EPR spectra challenging, but also their analysis. Comparison of the EPR results on the active site of desulforedoxin to those of the closely related active site of rubredoxin illustrates the necessity of explicit quantum-chemical calculations in order to interrelate the electronic and geometric structure of biological transition-metal sites.

Configuration of spheroidene in the photosynthetic reaction center of Rhodobacter sphaeroides : a comparison of wild-type and reconstituted R26.

We compare the resonance Raman spectra acquired at two excitation wavelengths, 496.5 and 514.5 nm, of spheroidene in the wild-type reaction center of Rhodobacter sphaeroides and reconstituted into the reaction center of the Rhodobacter sphaeroides mutant R26. Our earlier work showed that the reconstituted R26 reaction center holds spheroidene in two configurations: 15,15'-cis and another configuration. Here we show that in the wild-type reaction center only 15,15'-cis spheroidene is present. In the resonance Raman spectra of the reconstituted R26 reaction centers, a transition is identified that arises exclusively from the second configuration. According to density-functional-theory calculations, this transition is specific for the 13,14-cis configuration.

Action Spectroscopy on Dense Samples of Photosynthetic Reaction Centers of Rhodobacter sphaeroides WT Based on Nanosecond Laser-Flash C Photo-CIDNP MAS NMR.

Photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance (photo-CIDNP MAS NMR) allows for the investigation of the electronic structure of the photochemical machinery of photosynthetic reaction centers (RCs) at atomic resolution. For such experiments, either continuous radiation from white xenon lamps or green laser pulses are applied to optically dense samples. In order to explore their optical properties, optically thick samples of isolated and quinone-removed RCs of the purple bacteria of Rhodobacter sphaeroides wild type are studied by nanosecond laser-flash (13)C photo-CIDNP MAS NMR using excitation wavelengths between 720 and 940 nm. Action spectra of both the transient nuclear polarization as well as the nuclear hyperpolarization, remaining in the electronic ground state at the end of the photocycle, are obtained. It is shown that the signal intensity is limited by the amount of accessible RCs and that the different mechanisms of the photo-CIDNP production rely on the same photophysical origin, which is the photocycle induced by one single photon.

A Proton ENDOR Study of Azurin.

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cbeta atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein.

The electronic structure of the primary electron donor of reaction centers of purple bacteria at atomic resolution as observed by photo-CIDNP 13C NMR.

Composed of the two bacteriochlorophyll cofactors, P(L) and P(M), the special pair functions as the primary electron donor in bacterial reaction centers of purple bacteria of Rhodobacter sphaeroides. Under light absorption, an electron is transferred to a bacteriopheophytin and a radical pair is produced. The occurrence of the radical pair is linked to the production of enhanced nuclear polarization called photochemically induced dynamic nuclear polarization (photo-CIDNP). This effect can be used to study the electronic structure of the special pair at atomic resolution by detection of the strongly enhanced nuclear polarization with laser-flash photo-CIDNP magic-angle spinning NMR on the carotenoid-less mutant R26. In the electronic ground state, P(L) is strongly disturbed, carrying a slightly negative charge. In the radical cation state, the ratio of total electron spin densities between P(L) and P(M) is 2:1, although it is 2.5:1 for the pyrrole carbons, 2.2:1 for all porphyrinic carbons, and 4:1 for the pyrrole nitrogen. It is shown that the symmetry break between the electronic structures in the electronic ground state and in the radical cation state is an intrinsic property of the special pair supermolecule, which is particularly attributable to a modification of the structure of P(L). The significant difference in electron density distribution between the ground and radical cation states is explained by an electric polarization effect of the nearby histidine.

A W-band pulsed EPR/ENDOR study of Co(II)S(4) coordination in the Co[(SPPh(2))(SP(i)Pr(2))N](2) complex.

Spin-echo detection at 95 GHz enables an electron-paramagnetic-resonance study of a cobalt complex with a bio-mimetic coordination of the transition metal by four sulfur atoms. A magnetically diluted single crystal of the complex has been investigated in great detail. Electron-nuclear double-resonance signals were observed of ligand nuclei and complete hyperfine tensors of the distinct phosphorus nuclei were derived, assigned and discussed.

Multifrequency EPR analysis of the positive polaron in I2-doped poly(3-hexylthiophene) and in poly[2-methoxy-5-(3,7-dimethyloctyloxy)]-1,4-phenylenevinylene.

The W-band continuous-wave electron paramagnetic resonance (EPR) analysis of chemically induced polarons in drop-cast and spin-coated polyphenylenevinylene-type and polythiophene-type polymer films reveals rhombic g tensors in both cases. The dependence of the W-band EPR signals on the orientation of the spin-coated films with respect to the magnetic field indicates a high degree of backbone alignment with the substrate and allows a partial assignment of the g tensor orientation. The derived molecular orientations of the polymer chains in the spin-coated films show clear differences between the two types of polymers. The proton hyperfine interactions obtained from X-band HYSCORE (hyperfine sublevel correlation) and Q- and W-band pulsed ENDOR (electron-nuclear double resonance) experiments are interpreted in terms of earlier theoretical studies on the extension of the polarons.

Characterization of the primary radical pair in reaction centers of Heliobacillus mobilis by 13C photo-CIDNP MAS NMR.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by (13)C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the (13)C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 8(1)-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.

Signals in solid-state photochemically induced dynamic nuclear polarization recover faster than signals obtained with the longitudinal relaxation time.

During the photocycle of quinone-blocked photosynthetic reaction centers (RCs), photochemically induced dynamic nuclear polarization (photo-CIDNP) is produced by polarization transfer from the initially totally electron polarized electron pair and can be observed by 13C magic-angle spinning (MAS) NMR as a strong modification of signal intensities. The same processes creating net nuclear polarization open up light-dependent channels for polarization loss. This leads to coherent and incoherent enhanced signal recovery, in addition to the recovery due to light-independent longitudinal relaxation. Coherent mixing between electron and nuclear spin states due to pseudosecular hyperfine coupling within the radical pair state provides such a coherent loss channel for nuclear polarization. Another polarization transfer mechanism called differential relaxation, which is based on the long lifetime of the triplet state of the donor, provides an efficient incoherent relaxation path. In RCs of the purple bacterium Rhodobacter sphaeroides R26, the photochemical active channels allow for accelerated signal scanning by a factor of 5. Hence, photo-CIDNP MAS NMR provides the possibility to drive the NMR technique beyond the T1 limit.

13C chemical shift map of the active cofactors in photosynthetic reaction centers of Rhodobacter sphaeroides revealed by photo-CIDNP MAS NMR.

13C photo-CIDNP MAS NMR studies have been performed on reaction centers (RCs) of Rhodobacter sphaeroides wild type (WT) that have been selectively labeled with an isotope using [5-13C]-delta-aminolevulinic acid.HCl in all the BChl and BPhe cofactors at positions C-4, C-5, C-9, C-10, C-14, C-15, C-16, and C-20. 13C CP/MAS NMR and 13C-13C dipolar correlation photo-CIDNP MAS NMR provide a chemical shift map of the cofactors involved in the electron transfer process in the RC at the atomic scale. The 13C-13C dipolar correlation photo-CIDNP spectra reveal three strong components, originating from two BChl cofactors, called P1 and P2 and assigned to the special pair, as well as one BPhe, PhiA. In addition, there is a weak component observed that arises from a third BChl cofactor, denoted P3, which appears to originate from the accessory BChl BA. An almost complete set of assignments of all the aromatic carbon atoms in the macrocycles of BChl and BPhe is achieved in combination with previous photo-CIDNP studies on site-directed BChl/BPhe-labeled RCs [Schulten, E. A. M., Matysik, J., Alia, Kiihne, S., Raap, J., Lugtenburg, J., Gast, P., Hoff, A. J., and de Groot, H. J. M. (2002) Biochemistry 41, 8708-8717], allowing a comprehensive map of the ground-state electronic structure of the photochemically active cofactors to be constructed for the first time. The reasons for the anomaly of P2 and the origin of the polarization on P3 are discussed.