Catalase Activity of Cytochrome c Oxidase Assayed with Hydrogen Peroxide-Sensitive Electrode Microsensor

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I. A. Bolshakov¹, T. V. Vygodina², R. Gennis³, A. A. Karyakin¹, and A. A. Konstantinov^2*

¹Chemistry Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: konst@genebee.msu.ru

³School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801, USA

^* To whom correspondence should be addressed.

Received March 11, 2010; Revision received May 28, 2010

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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 oxidase 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 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.

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KEY WORDS: microsensor electrode, Prussian blue, respiratory chain, cytochrome c oxidase, oxygen intermediates, catalase activity

DOI: 10.1134/S0006297910110064

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