The heme vicinities of the acid and alkaline forms of native (Fe(III)) horse- radish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III) protoporphyrin IX dimethylester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentacoordinated heme complex. The MCD spectra of native horseradish peroxidase did not show any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin com- ponent of metmyoglobin hydroxide and metmyoglobin azide.
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