Haem propionates control oxidative and reductive activities of horseradish peroxidase by maintaining the correct orientation of the haem

Abstract

The role of haem propionates in oxidative and reductive reactions catalysed by horseradish peroxidase (HRP) was studied after successful reconstitution of ferric protoporphyrin IX dimethyl ester (PPDME) into the apoperoxidase. The reconstituted enzyme oxidizes neither guaiacol (aromatic electron donor) nor iodide or thiocyanate (inorganic donor). Although 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 spectroscopically detectable compound II with H₂O₂. CD measurements indicate a significant loss of haem CD spectrum of the reconstituted enzyme at 409 nm, suggesting a loss of asymmetry of the haem–protein interaction. Thus the inability of the reconstituted enzyme to form catalytic 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 of EDTA and H₂O₂. The reconstituted enzyme cannot catalyse I⁺ reduction because of the loss of I⁺ binding to the haem propionate. Since I⁺ reduction requires formation of the catalytically active enzyme–I⁺–EDTA ternary complex, the loss of reductive activity is primarily due to the 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 correct orientation of the haem with respect to the surrounding residues.