Coordination geometry of Cu-porphyrin in Cu(II)–Fe(II) hybrid hemoglobins studied by Q-band EPR and resonance Raman spectroscopies

Cu(II)–Fe(II) hybrid hemoglobins were investigated by UV–vis, Q-band (35 GHz) EPR and resonance Raman spectroscopies. EPR results indicated that Cu-porphyrin in α-subunit within hybrid hemoglobin had either 5- or 4-coordination geometry depending on the pH conditions, while Cu-porphyrin in β-subunit had only 5-coordination geometry at high and low pH values. These results were consistent with UV–vis absorption results. A new resonance Raman band appeared around 190 cm −1, which was present whenever 5-coordinated Cu-porphyrin existed in Cu(II)–Fe(II) hybrid hemoglobins irrespective of the coordination number in Fe(II) subunit. This Raman band might be assigned to Cu–N ϵ (His) stretching mode. These results are direct demonstration of the existence of coordination changes of Cu-porphyrin in α-subunit within hybrid hemoglobin by shifting the molecular conformation from fully unliganded state to intermediately liganded state.