title: The K(C) channel in the cbb3-type respiratory oxygen reductase from Rhodobacter capsulatus is required for both chemical and pumped protons.
abstract: The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome c oxidase as well as many bacterial homologues. Members of the C family, also called the cbb3-type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the K(C) channel) present in the cbb3-type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the K(C) channel of the cbb3-type oxygen reductase from Rhodobacter capsulatus were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the K(C) channel.
authors.affiliation: Department of Biology, Faculty of Literature and Science, Abant Izzet Baysal University, Bolu, Turkey.
authors.forename: Gülgez Gökçe
authors.initials: GG
authors.lastname: Yıldız
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
authors.forename: Mehmet
authors.initials: M
authors.lastname: Öztürk
mesh_terms: D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D016680:Genome, Bacterial; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption; D011487:Protein Conformation; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D017494:Proton Pumps; D010088:Oxidoreductases; D003576:Electron Transport Complex IV