A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins

Abstract

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.

High-frequency EPR applications of open nonradiative resonators

“…9 shows the ELDOR-detected NMR spectrum of the [RhCl 6 ] 4À complex in AgCl single crystal [41,42], obtained at 5 K for an orientation close to that of the largest principal g-value. The ELDOR-detected NMR experiments were performed using the pulse sequence (HTA) m 2 -t 1 -(p p=2 ) m 1 -s-(p p ) m1 -s-echo, based on rectangular pulses [43]. HTA indicates a high turning angle pulse with variable microwave frequency (m 2 ), whereas m 1 indicates a fixed microwave frequency.…”

Electron Paramagnetic Resonance and Mössbauer Spectroscopy and Density Functional Theory Analysis of a High-Spin FeIV–Oxo Complex

“…This effect is similar to the broadening observed in other water ligated ferric heme complexes. 36 The A x value and the sign of these values cannot be determined from the data. The dipolar contribution to the hyperfine tensor determined from DFT calculations was 170 A dip = (5.25, -0.35, -4.90) MHz.…”

Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM)

“…This is typical for pentacoordinated ferric iron in the high-spin state (S = 5/2) [17,18]. When the coordination symmetry of the complex changes from penta-to hexacoordination, the Fe(III) ion adopts the low-spin state (S = 1/2) yielding a signal centered around g ~2, if the sixth ligand is strongly coordinating [6,19].…”