Two- and four-pulse ESEEM studies of the heme binding center of a low-spin ferriheme protein: the importance of a multi-frequency approach

The histamine complex of nitrophorin 1 (NP1–Hm) was studied by ESEEM at X- and S-bands. The proton sum combination feature in the X-band ESEEM spectra was well resolved at the low-field g-value (gZ), but unresolved at the high-field g-value (gX). Decreasing the microwave frequency down to S-band allowed us to obtain well-resolved ESEEM spectra at the gX position and to determine the orientation of gX to be 60°–90° with respect to the plane of the axial ligands.