Equilibria between horseradish peroxidase and aromatic donors.

Equilibria between horseradish peroxidase and aromatic hydrogen donors have been analyzed spectrophotometrically and potentiometrically. The donors alter the peroxidase spectrum slightly but reproducibly with changes of two types. Donors of the two groups compete for the same binding site with no systematic difference in affinity for the enzyme. Donors with one aromatic ring are fairly loosely ligated, Kd3-25 mM, but enlargement, or extension of the pi-electron system, increases the affinity. A negative change in entropy and a large negative change in enthalpy upon binding indicates a specific donor-enzyme interaction, and the retention of the peroxidase by phenyl- but not by octyl-Sepharose points at the involvement of aromatic amino acid(s) in the ligation of an aromatic donor. Substitution of the hematin vinyl groups by ethyl or acetyl groups does not affect Kd of the peroxidase-donor complex. Reduction of the iron atom to Fe(II), or its removal, influences Kd only modestly. The fluorescence of the protoporphyrin-apoprotein HRP C2 associate is not quenched by donors from either group. These observations are in accord with NMR and other data from the literature and point at a ligation of the donor only to the protein moiety. Our results do not support the assumption of an Fe(III) H2O...donor hydrogen bond. The energy balance in the four-membered system free and donor-bound peroxidase Fe(III)/(II) has been analyzed. The model donors used in the present study modulate the redox properties only slightly. Plant peroxidases in situ may be donor-bound to a large extent.