Effect of quaternary structure change on the low-lying electronic states of the ferrous heme in deoxy-Hb studied by multi-frequency EPR.

The effect of the quaternary structure change in deoxy-Hb on the electronic states of the ferrous high-spin heme was studied by multi-frequency electron paramagnetic resonance (MFEPR). We used a single-pass transmission-type EPR at 70-220 GHz and at 1.5K. We compared the EPR spectra of deoxy-Hb at high pH with those at low pH and in the presence of the allosteric effectors, such as inositol hexaphosphate (IHP). Our EPR results imply that the low-lying electronic states of the ferrous heme in deoxy-Hb could be altered by the changes in the heme geometry, which might be caused by the quaternary structure change in Hb. We also measured EPR spectra of deoxy FeMg hybrid Hb to clarify the difference between α and β hemes at low pH with IHP. The additional complicated resonance branches appeared in deoxy-Hb as well as in deoxy FeMg hybrid Hbs at low pH with IHP may reflect the energy states of the ferrous high-spin heme correlated with the low oxygen affinity state. The magnetic moment of the lowest excited state of the α heme seems to be different from that of the β heme in the low oxygen affinity state.