title: Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase.
abstract: Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.
authors.affiliation: Johann Wolfgang Goethe-Universität, Biozentrum, Institut für Biochemie, Molekulare Genetik, Frankfurt, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Pfitzner
authors.forename: A
authors.initials: A
authors.lastname: Kirichenko
authors.forename: A A
authors.initials: AA
authors.lastname: Konstantinov
authors.forename: M
authors.initials: M
authors.lastname: Mertens
authors.forename: A
authors.initials: A
authors.lastname: Wittershagen
authors.forename: B O
authors.initials: BO
authors.lastname: Kolbesen
authors.forename: G C
authors.initials: GC
authors.lastname: Steffens
authors.forename: A
authors.initials: A
authors.lastname: Harrenga
authors.forename: H
authors.initials: H
authors.lastname: Michel
authors.forename: B
authors.initials: B
authors.lastname: Ludwig
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003300:Copper / Q000378:metabolism; D018360:Crystallography, X-Ray; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D009154:Mutation; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D013052:Spectrometry, X-Ray Emission; D014867:Water
chemical_list: D014867:Water; D003300:Copper; D012964:Sodium; D007501:Iron; D003576:Electron Transport Complex IV; D002118:Calcium