Characterization of a de novo designed heme protein by EPR and ENDOR spectroscopy

Abstract

The binding situation of heme incorporated into a de novo synthesized protein is investigated with EPR and ENDOR spectroscopy. The protein was modeled on the cytochrome b subunit of the cytochrome bc1 complex and contains two bis-histidine heme binding sites. The EPR spectra show Fe3+ low-spin signals with g tensor principal values of 2.97, 2.27, and 1.51 and a contribution of a highly anisotropic low-spin (HALS) species with a gmax signal at g = 3.5. The regular Fe3+ low-spin EPR spectra were simulated based on a g strain linewidth-broadening mechanism. The resulting g tensor principal values were used for a ligand-field analysis. The ligand-field parameters are typical for bis-histidine ligated hemes with approximately parallel histidine planes. The HALS species is characteristic of bis-histidine ligation with tilted or twisted imidazole planes. The occurrence of both types of heme in the de novo synthesized protein is discussed. The EPR data are supplemented by pulsed ENDOR studies of this protein and are compared with those of metmyoglobin-imidazole and bis-imidazole ferric heme model systems. ENDOR resonances of nitrogen and protons of histidine were identified and used as structural probes for the axial ligation of the hemes.