title: True wild type and recombinant wild type cytochrome c oxidase from Paracoccus denitrificans show a 20-fold difference in their catalase activity.
abstract: The four subunit (SU) aa(3) cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. Its binuclear active center, residing in SU I, contains heme a(3) and Cu(B). Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity. To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT strain was constructed, carrying the gene for SU I on a low copy number plasmid. This rec. WT showed no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential over-production of SU I might impair the correct insertion of heme a(3) and Cu(B) because of a deficiency in metal inserting chaperones. An altered distance between heme a(3) and Cu(B) and variations in protein structure are possible reasons for the observed increased catalase activity. The availability of chaperones was improved by cloning the genes ctaG and surf1c on the same plasmid as the SU I gene. The new rec. WT CcO showed in fact a reduced catalase activity. Using differential scanning calorimetry no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging the thermal stability of the rec. WT CcO was reduced compared to that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.
authors.affiliation: Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany. Florian.Hilbers@biophys.mpg.de
authors.forename: Florian
authors.initials: F
authors.lastname: Hilbers
authors.forename: Iris
authors.initials: I
authors.lastname: von der Hocht
authors.forename: Bernd
authors.initials: B
authors.lastname: Ludwig
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D002152:Calorimetry, Differential Scanning; D002374:Catalase / Q000378:metabolism*; D003001:Cloning, Molecular; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002374:Catalase; D003576:Electron Transport Complex IV