title: Electron transfer between cytochrome c and the isolated CuA domain: identification of substrate-binding residues in cytochrome c oxidase.
abstract: Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Lappalainen
authors.forename: N J
authors.initials: NJ
authors.lastname: Watmough
authors.forename: C
authors.initials: C
authors.lastname: Greenwood
authors.forename: M
authors.initials: M
authors.lastname: Saraste
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D003574:Cytochrome c Group; D011994:Recombinant Proteins; D003300:Copper; C031732:cytochrome C-550; D003576:Electron Transport Complex IV