Determination of zero-field splitting and evidence for the presence of charge-transfer transitions in the Soret region of high-spin ferric hemoproteins obtained from an analysis of low-temperature magnetic circular dichroism

Theoretical expressions for magnetic circular dichroism (MCD) of the porphyrin π→π*, spin-allowed charge transfer (CT) and spin-forbidden d-d or CT transitions in high-spin ferric heme are derived. The transitions can be discriminated by their MCD to absorption ratio and/or temperature dependence of MCD intensity. An analysis of the Soret MCD of fluoride complexes of myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), recorded at temperatures from 290 down to 2 K, is given. It is shown that the Soret MCD of HRPF can be well described by overlapping of the π→π* transition with one spin-forbidden CT transition of an ⁶A₁→⁴E type. In the case of MbF and HbF it is necessary to assume the presence in the Soret region of the second spin-forbidden CT transition, most probably of an ⁶A₁→⁴A₁ type. The parameters of transitions have been extracted from a non-linear least-squares fitting procedure. The best fit values of parameter D of the zero-field splitting of the ground manifold for HbF (6.1 cm^-1) and MbF (6.4 cm^-1) agree well with those obtained by other methods. The D value for HRPF (8.3 cm^-1) is obtained for the first time.