Multifrequency EPR Study of Fe3+ and Co2+ in the Active Site of Desulforedoxin

The understanding of the electronic structure of S > 1/2 transition-metal sites that show a large zero-field splitting (ZFS) of the magnetic sublevels benefits greatly from study by electron-paramagnetic-resonance (EPR) spectroscopy at frequencies above the standard 9.5 GHz. However, high-frequency EPR spectroscopy is technically challenging and still developing. Particularly the sensitivity of high-frequency EPR spectrometers is often too low to apply the technique in the study of transition-metal sites in proteins and enzymes. Here we report a multifrequency EPR study (at 9.5, 94.9, and 275.7 GHz) of the active site of the protein desulforedoxin, both in its natural Fe3+ form and substituted with Co2+. The 275.7 GHz EPR spectra made it possible to determine the ZFS parameters of the Fe3+ site with high precision. No 275.7 GHz spectrum could be observed of the Co2+ site, but based on 9.5 GHz spectra, its ZFS parameters could be estimated. We find that the typical variation in the geometry of the active site of a protein or enzyme, referred to as conformational strain, does not only make the detection of EPR spectra challenging, but also their analysis. Comparison of the EPR results on the active site of desulforedoxin to those of the closely related active site of rubredoxin illustrates the necessity of explicit quantum-chemical calculations in order to interrelate the electronic and geometric structure of biological transition-metal sites.