Zero-field splittings in metHb and metMb with aquo and fluoro ligands: a FD-FT THz-EPR study

Abstract

A combined X-band and frequency-domain Fourier-transform THz electron paramagnetic resonance (FD-FT THz-EPR) approach has been employed to determine heme Fe(III) S = 5/2 zero-field splitting (ZFS) parameters of frozen metHb and metMb solutions, both with fluoro and aquo ligands. Frequency-domain EPR measurements have been carried out by an improved synchrotron-based FD-FT THz-EPR spectrometer. ZFS has been determined by field dependence of spin transitions within the m_S = ±1/2 manifold, for all four protein systems, and by zero-field spin transitions between m_S = ±1/2 and m_S = ±3/2 levels, for metHb and metMb flouro-states. FD-FT THz-EPR data were simulated with a novel numerical routine based on Easyspin, which allows now for direct comparison of EPR spectra in field and frequency domain. We found purely axial ZFSs of D = 5.0(1) cm⁻¹ (flouro-metMb), D = 9.2(4) cm⁻¹ (aquo-metMb), D = 5.1(1) cm⁻¹ (flouro-metHB) and D = 10.4(2) cm⁻¹ (aquo-metHb).

Keywords:

• haemoglobin
• myoglobin
• EPR spectroscopy
• zero-field splitting

Acknowledgements

This work has been supported and funded by Deutsche Forschungsgemeinschaft (DFG) priority program SPP 1601. B.M. Martins is supported by DFG (MA 3348/2-2). FD-FT THz-EPR has been carried out within the BESSY II user program. We thank Sun Un (CEA, Saclay) for helpful discussions and Dirk Ponwitz (HZB) for technical support.