Analysis of the EPR spectra of transferrin: the importance of a zero-field-splitting distribution and 4th-order terms

Abstract

Multi-frequency EPR spectroscopy can provide high-level structural information on high-spin Fe³⁺ sites in proteins and enzymes. Unfortunately, analysis of the EPR spectra of these spin systems is hindered by the presence of broad distributions in the zero-field-splitting (ZFS) parameters, which reflect conformational heterogeneity of the iron sites. We present the analysis of EPR spectra of high-spin Fe³⁺ bound to human serum transferrin. We apply a method termed the grid-of-errors to extract the distributions of the individual ZFS parameters from EPR spectra recorded in the high-field limit at a microwave frequency of 275 GHz. Study of a series of transferrin variants shows that the ZFS distributions are as characteristic of the structure of a high-spin Fe³⁺ site as the ZFS parameters themselves. Simulations based on the extracted ZFS distributions reproduce spectra recorded at 34 GHz (Q band) and 9.7 GHz (X band), including subtle variations that were previously difficult to quantify. The X-band spectrum of transferrin shows a characteristic double peak, which has puzzled researchers for decades. We show that the double peak is uniquely related to the term B₄⁻³O₄⁻³(S) in the spin Hamiltonian. Our method is generally applicable in the analysis of spectra that arise from a broad distribution of parameters.