Heme-linked ionization in compounds I and II of horseradish peroxidases A2 and C.

Heme-linked ionizations in Compound I and II of horseradish peroxidases, the presence of which was suggested from kinetic data by H. B. Dunford and J. S. Stillman [(1976) Coordination Chem. Rev.19, 187], were detected from two independent experiments of spectrophotometric titration and proton balance. The values of pK_a in Compound II were 6.9 for peroxidase A₂ and 8.5 for peroxidase C. The kinetic results were accounted for by assuming that the alkaline forms of Compound II are inactive or very sluggish toward electron donors. It was concluded that the two ionizations occur in a functionally homologous position of the two isoenzymes, which is the distal group itself or closely related to it. A heme-linked ionization of pK_a = ca. 5.4 in Compound I of peroxidase C could be detected from pH changes of the visible spectrum. Measuring proton balance in each step of reductions from Compound I to Compound II to the ferric enzyme, it was found that ionizations having similar pK_a values of 5.1–5.4 are present in both Compound I and the ferric enzyme. The pK_a group in the ferric enzyme was confirmed to correspond with that reported by H. Theorell and K. G. Paul [(1944) Arkiv Kemi Mineral. Geol.18A, No. 12]. A tentative model for the vicinity of heme-iron of peroxidase C is presented as a working hypothesis.