S. Van Doorslaer

A multi-frequency pulse EPR and ENDOR approach to study strongly coupled nuclei in frozen solutions of high-spin ferric heme proteins.

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Analyzing heme proteins using EPR techniques: the heme-pocket structure of ferric mouse neuroglobin

In this work, an electron paramagnetic resonance (EPR) strategy to study the heme-pocket structure of low-spin ferric heme proteins is optimized. Frozen solutions of ferric mouse neuroglobin (mNgb) are analyzed by means of electron spin echo envelope modulation and pulsed electron–nuclear double resonance techniques. The hyperfine and nuclear quadrupole couplings of the directly coordinating heme and histidine nitrogens are derived and are discussed in comparison with known data of other ferric porphyrin compounds. In combination with the hyperfine matrices of the imidazole protons, the 14N EPR parameters reveal structural information on the heme pocket of mNgb that is in agreement with previous X-ray diffraction data on neuroglobins.

Analysing low-spin ferric complexes using pulse EPR techniques: a structure determination of bis (4-methylimidazole)(tetraphenylporphyrinato)iron(III)

Continuous-wave (CW) EPR has been extensively used as a characterization tool for low-spin ferric complexes. Using the bis(4-methylimidazole) complex of iron(III) tetraphenylporphyrin as an example we show how the combination of CW EPR and pulsed EPR techniques allows for a detailed structure analysis of such ferric complexes. Both proton HYSCORE and combination-peak experiments indicate that the imidazole ligand planes are (nearly) parallel to the gx axis (±20°). Simulations of the porphyrin nitrogen contributions to the HYSCORE spectra allow a determination of the g-axes frame in the molecular frame. Combination of these spectral results and the counter-rotation principle show that the gx and gy axes are rotated 5° (±5°) away from the Np–Fe–Np axes and that the imidazole ligand planes are counter-rotated over 5° (±5°). The nitrogen hyperfine and nuclear-quadrupole interactions of the pyrrole and imidazole nitrogens are determined and discussed as a function of the electronic structure.

EPR-spectroscopic evidence of a dominant His-FeIII-His coordination in ferric neuroglobin

Abstract The ferric form of the wild-type mouse neuroglobin (Ngb), a newly discovered heme protein which is primarily expressed in the brain of mammals, has been characterized by electron paramagnetic resonance (EPR) spectroscopy. The study reveals the simultaneous presence of two related structural forms in a wide range of pH values. The dominant low-spin form (>90%) with g -tensor principal values 3.15, 2.16 and 1.34 can be attributed to a His–Fe III –His configuration. The high-spin form with g ⊥ =5.97 and g ∥ ∼2, can be ascribed either to a hexacoordinated His–Fe III –H 2 O form or to a pentacoordinated His–Fe III . The high-spin to low-spin ratio is found to decrease with increasing pH values.

Distance determination between low-spin ferric haem and nitroxide spin label using DEER: the neuroglobin case

This work demonstrates for the first time the feasibility of using double electron–electron resonance (DEER) to determine the inter-spin distance between nitroxide spin labels and low-spin (S = 1/2) ferric haem centres. For these means, two human neuroglobin variants were spin labelled leading to singly labelled haem proteins with the nitroxide label on one of the natural Cys residues (Cys55 or Cys120). Room-temperature electron paramagnetic resonance was used to characterise the mobility of the nitroxide labels and X- and Q-band DEER experiments were performed to detect nitroxide–haem distances. Effects of residual nuclear modulations in the DEER traces were carefully evaluated. The DEER-derived distances were compared with theoretical predictions from an X-ray diffraction structure of human neuroglobin using a rotamer library approach as well as with distance information obtained from electron relaxation measurements. The structural biological implications of the spin-labelled side chains’ dynamics and of the obtained distances are also discussed.

Studying High-Spin Ferric Heme Proteins by Pulsed EPR Spectroscopy: Analysis of the Ferric Form of the E7Q Mutant of Human Neuroglobin

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave electron paramagnetic resonance (CW EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. It is shown that the use of matched pulses in the HYSCORE experiment is essential to observe the nitrogen spectral contributions. The validity of approximating the high-spin Fe(III) system (S=5/2) as an effectiveS=1/2 system is tested and the consequences for the HYSCORE simulations are highlighted. Comparative HYSCORE experiments combined with deuterium exchange experiments for aquometmyoglobin and ferric E7Q-NGB clearly show that the heme iron of the latter protein is pentacoordinated, lacking the distal water. Furthermore, CW EPR experiments show that, at high pH, the E10K residue is coordinating to the heme iron in this globin. These observations are corroborated by resonance Raman experiments and could also be reproduced for other E7 mutants of human and mouse neuroglobin. Finally, the proton and nitrogen hyperfine and nuclear quadrupole parameters obtained for ferric E7Q-NGB are discussed in detail.

31P and1H powder ENDOR study of ozonide radicals in carbonated apatites, synthesized from aqueous solutions

An O3− defect in Na+ CO32− containing apatite powder has been investigated with ENDOR after X-irradiation. The powder, synthesized by a hydrolysis of octo-calciumphosphate (OCP) and Na2CO3 was dried at 25°C until constant weight was reached. At low temperatures, both31P and1H ENDOR spectra were recorded for different settings of the magnetic field (i.e., when the magnetic field is swept through the EPR O3− spectrum). The ENDOR powder spectra were thoroughly analyzed using computer simulations based on the “orientation selection principle”. Interactions with two types of protons and two types of31P nuclei could be resolved. In this way, a detailed model could be established for the O3− ion in the hydroxyapatite lattice. The defect is located between two successive vacant hydroxyl sites. The axis connecting the two outer oxygen atoms (gy-axis) of the O3− ion is found to be along the hexagonalc-axis of the lattice. The twofold axis of the defect ion (gz-axis) is parallel to theb-axis of the lattice.

Ligand Binding to Chlorite Dismutase from Magnetospirillum Sp.

Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.

Evaluating π-π stacking effects in macrocyclic transition metal complexes using EPR techniques

The HYSCORE spectra for two different macrocyclic transition metal complexes, namely cobalt tetraphenylporphyrin (CoTPP) and a copper Salen derivative ([Cu(1)]), were examined in relation to their interactions with pyridine (Py) and methylbenzyl amine (MBA), respectively. In both cases weak hyperfine interactions were detected by HYSCORE, but the origin of these interactions was found to originate from completely different effects. In the CoTPPpy adduct, with axial coordination of the substrate (pyridine) to the metal centre, weak couplings from the pyrrole nitrogens of a neighbouring porphyrin complex were identified, and confirmed through a series of isotopic labelling and dilution experiments. This result represents the first ever identification by HYSCORE of π-π interactions between such porphyrin complexes in solution. In the [Cu(1)]MBA adduct, again with axial coordination of the substrate (MBA) to the copper centre, weak couplings were also identified in the HYSCORE spectra, which could easily be misinterpreted as arising from intermolecular interactions between adjacent ligands. However, the origin of these couplings was clearly demonstrated to arise from intramolecular 13C-ligand interactions. These results demonstrate not only the sensitivity of the HYSCORE technique for detection of weak inter- and intra-molecular interactions in macrocyclic transition metal complexes, but additionally the need to consider dilution effects in the spectral assignments.

Structural analysis of newly designed platinum compounds with interesting conductivity and optical properties

In the past years a great effort has been put in the design and synthesis of stable processable materials with metallic or semi-conducting properties. In order to fully exploit the possibilities of a newly synthesized conducting material its structure-function relation needs to be unraveled. Using two specific examples, it will be shown that modern EPR can reveal interesting properties of conducting materials. A detailed analysis will be given of a series of novel soluble platinum compounds designed to mimic the Magnus salt characteristics: [PtL2][Pt(mnt)2] and [PtL2][Pt(dmit)2], where L represents 1,10-phenanthroline or 4,4-dimethyl-2,2′-dipyridyl, and mnt (dmit) denotes 1,2-dicyanoethylenedithiolate (1,3-dithiol-2-thione-4,5-dithiolate). The studied complexes have semiconductive properties and high thermal stability. Although chemical analysis predicted the compounds to be diamagnetic, cw EPR revealed the presence of a considerable amount of paramagnetic species in the compounds. On the basis of a cw EPR and HYSCORE study at X-band, the species could be fully identified and the relation to the conductivity properties of the samples will be discussed.