A. Schweiger

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.

Solvent effects of cobalt(II) phthalocyanine in sulfuric acid: a continuous wave and pulse EPR study

Products based on metal phthalocyanines are widely used in industry. In the processing of these materials it is essential to control the conditions of the matrix the metal phthalocyanines are embedded in. Using the example of cobalt(II) phthalocyanine in sulfuric acid we show that continuous wave and pulse electron paramagnetic resonance and electron nuclear double resonance spectroscopy provide excellent tools to monitor the influence of the matrix on paramagnetic phthalocyanines. The g values, the cobalt hyperfine values, and the hyperfine and nuclear quadrupole couplings of the isoindole nitrogens, as well as the hyperfine interactions of the surrounding protons allow for a detailed assessment of the electronic structure of cobalt(II) phthalocyanine in sulfuric acid. Subtle differences between the system under study and related Co(II) porphyrin and corrin systems could be traced.

Numerical simulation of ESEEM experiments

Numerical simulation has become an indispensable tool for the interpretation of pulse EPR experiments. In this work it is shown how automatic orientation selection, grouping of operator factors, and direct selection and elimination of coherences can be used to improve the efficiency of time-domain simulations of one- and two-dimensional electron spin echo envelope modulation (ESEEM) spectra. The program allows for the computation of magnetic interactions of any symmetry and can be used to simulate spin systems with an arbitrary number of nuclei with any spin quantum number. Experimental restrictions due to finite microwave pulse lengths are addressed and the enhancement of forbidden coherences by microwave pulse matching is illustrated. A comparison of simulated and experimental HYSCORE (hyperfine sublevel correlation) spectra of ordered and disordered systems with varying complexity shows good qualitative agreement.