The chemical nature of the second hydrogen peroxide compound formed by cytochrome c peroxidase and horseradish peroxidase. 2. Formation and decomposition

Abstract

Spectrophotometric titrations of horseradish peroxidase and cytochrome c peroxidase with hydrogen peroxide suggest that more than 1 mole of compound II (2d intermediate compound formed by reaction of H2O2 with cytochrome c or horseradish peroxidase) can be formed for 1 mole of hydrogen peroxide. Further expts. established that compound II is formed in a series of irreversible reactions competing for the oxidizing capacity of the peroxide. Reducing groups present in the enzyme system play an essential role in these reactions. Compound II cannot dissociate into ferriperoxidase since its Fe atom has an effective oxidation number of +4: only reduction can regenerate ferriperoxidase. This process, which occurs spontaneously with trace-reducing matter present in the enzyme sample, is more rapid if sodium fluoride or potassium cyanide are added and the fluoride or cyanide complex of ferriperoxidase is the end product. There appears to be some correlation between the velocity constant for this enhanced reduction and the equilibrium constant for the fluoride or cyanide complex formation. If a slight excess of peroxide is added to the fluoride complex, compound II is formed and then disappears with reformation of the fluoride complex. With a larger excess of peroxide some compound I is also formed. The way in which this reaction occurs excludes an explanation based on shifting the position of an equilibrium in the system; instead, peroxide must be used up in irreversible reactions. The peroxide reactions of met-myoglobin, peroxidase and catalase are discussed in terms of the redox properties of the intermediate compounds and the way they are formed.