Structure and spin density of ferric low-spin heme complexes determined with high-resolution ESEEM experiments at 35 GHz
The wide use of the heme group by nature is a consequence of its unusual “electronic flexibility.” Major changes in the electronic structure of this molecule can result from small perturbations in its environment. To understand the way the electronic distribution is dictated by the structure of the heme site, it is extremely important to have methods to reliably determine both of them. In this work we propose a way to obtain this information in ferric low-spin heme centers via the determination of g, A, and Q tensors of the coordinated nitrogens using electron spin echo envelope modulation experiments at Q-band microwave frequencies. The results for two bisimidazole heme model complexes, namely, PPIX(Im)2 and CPIII(Im)2, where PPIX is protoporphyrin IX, CPIII is coproporphyrin III, and Im is imidazole, selectively labeled with 15N on the heme or imidazole nitrogens are presented. The planes of the axial ligands were found to be parallel and oriented approximately along one of the N–Fe–N directions of the slightly ruffled porphyrin ring (approximately 10°). The spin density was determined to reside in an iron d orbital perpendicular to the heme plane and oriented along the other porphyrin N–Fe–N direction, perpendicular to the axial imidazoles. The benefit of the method presented here lies in the use of Q-band microwave frequencies, which improves the orientation selection, results in no/fewer combination lines in the spectra, and allows separation of the contributions of hyperfine and quadrupole interactions due to the fulfillment of the exact cancellation condition at g Z and the possibility of performing hyperfine decoupling experiments at the g X observer position. These experimental advantages make the interpretation of the spectra straightforward, which results in precise and reliable determination of the structure and spin distribution.
KeywordsLow-spin heme Hyperfine sublevel correlation spectroscopy Heme protein Electron structure Ferric iron
Deadtime-free electron spin echo envelope modulation nuclear coherence-transfer echoes
Electron–nuclear double resonance
Electron paramagnetic resonance
Electron spin echo envelope modulation
Hyperfine sublevel correlation spectroscopy
This paper is dedicated to the memory of Arthur Schweiger, who could not see this work completed. The authors want to thank ETH Zürich and the Swiss National Science Foundation for financial support.
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