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

I. A. Bolshakov, T. V. Vygodina, Robert B Gennis, A. A. Karyakin, A. A. Konstantinov

Research output: Contribution to journalArticle

Abstract

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 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/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.

Original languageEnglish (US)
Pages (from-to)1352-1360
Number of pages9
JournalBiochemistry (Moscow)
Volume75
Issue number11
DOIs
StatePublished - Nov 1 2010

Fingerprint

Microsensors
Electron Transport Complex IV
Catalase
Hydrogen Peroxide
Electrodes
Rate constants
Oxidoreductases
Enzymes
Peroxides
Oxygen
Decomposition
Rhodobacter sphaeroides
Microelectrodes
Cytochromes
Superoxides
Transition metals
Metal ions
Hot Temperature
Metals
Ions

Keywords

  • Catalase activity
  • Cytochrome c oxidase
  • Microsensor electrode
  • Oxygen intermediates
  • Prussian blue
  • Respiratory chain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Catalase activity of cytochrome c oxidase assayed with hydrogen peroxide-sensitive electrode microsensor. / Bolshakov, I. A.; Vygodina, T. V.; Gennis, Robert B; Karyakin, A. A.; Konstantinov, A. A.

In: Biochemistry (Moscow), Vol. 75, No. 11, 01.11.2010, p. 1352-1360.

Research output: Contribution to journalArticle

Bolshakov, I. A. ; Vygodina, T. V. ; Gennis, Robert B ; Karyakin, A. A. ; Konstantinov, A. A. / Catalase activity of cytochrome c oxidase assayed with hydrogen peroxide-sensitive electrode microsensor. In: Biochemistry (Moscow). 2010 ; Vol. 75, No. 11. pp. 1352-1360.
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AU - Konstantinov, A. A.

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