HEME PROPIONATES CONTROL OXIDATIVE AND REDUCTIVE ACTIVITIES OF HORSERADISH-PEROXIDASE BY MAINTAINING THE CORRECT ORIENTATION OF THE HEME

The role of haem propionates in oxidative and reductive reactions cata lysed by horseradish peroxidase (HRP) was studied after successful rec onstitution of ferric protoporphyrin IX dimethyl ester (PPDME) into th e apoperoxidase. The reconstituted enzyme oxidizes neither guaiacol (a romatic electron donor) nor iodide or thiocyanate (inorganic donor). A lthough the reconstituted enzyme binds guaiacol with a similar K-d (13 mM) to that of the native enzyme (10 mM), the K-d for SCN- binding (5 mM) is decreased 20-fold compared with that of the native enzyme (100 mM). This indicates that haem propionates hinder the entry or binding of inorganic anion to the active site of the native HRP. However, the reconstituted enzyme is catalytically inactive as it does not form sp ectroscopically detectable compound II with H2O2. CD measurements indi cate a significant loss of haem CD spectrum of the reconstituted enzym e at 409 nm, suggesting a loss of asymmetry of the haem-protein intera ction. Thus the inability of the reconstituted enzyme to form catalyti c intermediates results from the change in orientation of the haem due to loss of interactions via the haem propionates. HRP also catalyses reductive reactions such as reduction of iodine (I+) in the presence o f EDTA and H2O2. The reconstituted enzyme cannot catalyse Ic reduction because of the loss of I+ binding to the haem propionate. Since I+ re duction requires formation of the catalytically active enzyme-I+-EDTA ternary complex, the loss of reductive activity is primarily due to th e loss of active enzyme formation. Haem propionates thus play a vital role in the oxidative and reductive reactions of HRP by favouring the formation of catalytic intermediates with H2O2 by maintaining the corr ect orientation of the haem with respect to the surrounding residues.