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 H2O2-dependent conversion of the oxidase intermediate FI-607 to FII-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/CuB 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.