title: Catalase activity of cytochrome C oxidase assayed with hydrogen peroxide-sensitive electrode microsensor.
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 oxidase is characterized by a second order rate constant of ~2·10(2) 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 a(3)/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.
authors.affiliation: Chemistry Faculty, Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: I A
authors.initials: IA
authors.lastname: Bolshakov
authors.forename: T V
authors.initials: TV
authors.lastname: Vygodina
authors.forename: R
authors.initials: R
authors.lastname: Gennis
authors.forename: A A
authors.initials: AA
authors.lastname: Karyakin
authors.forename: A A
authors.initials: AA
authors.lastname: Konstantinov
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002138:Calibration; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology; D009154:Mutation; D009206:Myocardium / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013045:Species Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV