Hydrogen peroxide decomposition by aa3 cytochrome c oxidase from Paracoccus denitrificans

The four subunit (SU) aa₃ cytochrome c oxidase from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain, reducing molecular oxygen to water. Its binuclear active center, residing in SU I, is composed of heme a₃ and Cu_B, the latter being ligated by three histidine residues. Apart from its main oxidoreductase activity, the protein shows a peroxidase (Orii, 1982) and a catalase activity (Orii, and Okunuki, 1963). To compare variants and the wild type (WT) protein in a more systematic way, a recombinant (rec.) WT was contructed, carrying the gene for SU I on a low copy number plasmid. This rec. WT showed, as expected, no difference in cytochrome c oxidase activity compared to the American type culture collection (ATCC) WT but surprisingly the k_cat of the catalase reaction was increased twentyfold. An altered distance of heme a₃ and Cu_B due to a slight overexpression of SU I and resulting variations in protein structure are a potential reason for the observed increase of the catalase activity. The slight overexpression of SU I might impair the correct insertion of the redox active metal centres due to a deficiency in metal inserting chaperones. The availability of chaperones was improved by cloning the genes for ctaG (Greiner et al., 2008) and surfIc (Bundschuh et al., 2008) on the same plasmid as SU I which indeed reduced the k_cat of the catalase reaction of the recombinant wild type. During the reaction of catalases two molecules of hydrogen peroxide are disproportioned to one molecule of oxygen and two water molecules. This reaction comprises a porphyrin radical (Compound I) (Fita and Rossmann, 1985), whereby the produced oxygen molecule originates from one hydrogen peroxide molecule (Jarnagin and Wang, 1953). The mechanism of hydrogen peroxide decomposition by cytochrome c oxidase was analyzed. Catalases accomplish the decomposition of hydrogen peroxide at their heme moiety; to investigate whether Cu_B is necessary for the catalase activity of cytochrome c oxidase, we constructed variants missing Cu_B. These variants possessed considerable catalase activity. Measurements using inhibitors showed that reduced iron (Fe²⁺) is not involved in the catalase reaction of cytochrome c oxidase and confirmed that Cu_B is dispensable for the dismutation of hydrogen peroxide by cytochrome c oxidase.