A 4-term energy level scheme for the high-spin ferrous hemoproteins: Evidence for the ⁵E_η and ⁵B₂ terms as the ground multiplets in hemoproteins with a histidine and a cysteine protein-derived heme ligand, respectively

We have carried out analysis of the electronic level scheme of the high-spin ferrous hemoproteins by simultaneous fit of the adjustable parameters of a 4-term theoretical model to low-temperature magnetic circular dichroism (MCD), room temperature absorption spectra and available magnetic susceptibility and/or Mössbauer data of myoglobin, horseradish peroxidase and cytochrome P450. The high reliability of the ligand field parameter values obtained for deoxymyoglobin is confirmed by good agreement between the predicted and observed magnetic field dependences of MCD and magnetization not used in the fit procedure. In addition, an energy gap between the ground and first excited singlets, estimated to be 4.2 cm^-1, agrees well with the value of ∼4 cm^-1 derived from the far-infrared magnetic resonance. Our computer and explicit theoretical analyses give strong evidence that large distinctions in the shape, intensity and temperature behaviour of the MCD of Mb and HRP from those of cytochrome P450 can be described only if the ground manifold in these proteins is ⁵E_η and ⁵B₂, respectively. The changes in relative energies of the one-electron 3d-orbitals on substitution of an imidazole of histidine for a sulphur anion of cysteine as a protein-derived heme iron ligand are rationalized by the lower ionization potential of the negatively charged sulphur ligand and the higher π-orbital overlap of its lone pair orbitals with the iron d_π-orbitals compared to the imidazole ligand.