jp709854x_si_002.pdf (1.94 MB)
A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins
journal contribution
posted on 2008-03-27, 00:00 authored by M. Fittipaldi, I. García-Rubio, F. Trandafir, I. Gromov, A. Schweiger, A. Bouwen, S. Van DoorslaerIn 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.