Equilibria between Horseradish Peroxidase and Aromatic Donors.

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Abstract
Equilibria between horseradish peroxidase (HRP) and aromatic hydrogen donors were analyzed spectrophotometrically and potentiometrically. The donors alter the peroxidase spectrum slightly but reproducibly with changes of 2 types. Donors of the 2 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, Kd 3-25 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 Fe atom to Fe(II), or its removal, influences Kd only modestly. The fluorescence of the protoporphyrin .cntdot. 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. The assumption of an Fe(III) H2O donor H bond was not supported. The energy balance in the 4-membered system free and donor-bound peroxidase Fe(III)/(II) was 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 great extent.

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