Catalase activity of cytochrome c oxidase assayed with hydrogen peroxide-sensitive electrode microsensor

An iron-hexacyanide-covered microelectrode sensor has been used to continuously monitor the kinetics of hydrogen peroxide decomposition catalyzed by oxidized cytochrome oxidase. At cytochrome oxidase concentration ̃1 μM, the catalase activity behaves as a first order process with respect to peroxide at concentrations up to ̃300-400 μM and is fully blocked by heat inactivation of the enzyme. The catalase (or, rather, pseudocatalase) activity of bovine cytochrome oxi-dase is characterized by a second order rate constant of ̃2.10² M^-1. sec-1 at pH 7.0 and room temperature, which, when divided by the number of H₂O₂ molecules disappearing in one catalytic turnover (between 2 and 3), agrees reasonably well with the second order rate constant for H₂O ₂dependent conversion of the oxidase intermediate F_I-607 to F_II-580. Accordingly, the catalase activity of bovine oxidase may be explained by H₂O₂ procession in the oxygen-reducing center of the enzyme yielding superoxide radicals. Much higher specific rates of H₂O₂ decomposition are observed with preparations of the bacterial cytochrome c oxidase from Rhodobacter sphaeroides. The observed second order rate constants (up to ̃3000 M^-1.sec^-1) exceed the rate constant of peroxide binding with the oxygen-reducing center of the oxidized enzyme (̃500 M^-1.sec^-1) several-fold and therefore cannot be explained by catalytic reaction in the a₃/Cu _B site of the enzyme. It is proposed that in the bacterial oxidase, H₂O₂ can be decomposed by reacting with the adventitious transition metal ions bound by the polyhistidine-tag present in the enzyme, or by virtue of reaction with the tightly-bound Mn²⁺, which in the bacterial enzyme substitutes for Mg²⁺ present in the mitochondrial oxidase.