Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase

FEBS Letters

Mertens, Martina⁵  Kolbesen, Bernd O.⁵  Pfitzner, Ute¹  Harrenga, Axel⁴  Wittershagen, Axel⁵  Kirichenko, Anna²  Michel, Hartmut⁴  Ludwig, Bernd¹  Steffens, Guy C.M.³  Konstantinov, Alexander A.² 

[1] Johann Wolfgang Goethe-Universität, Biozentrum, Institut für Biochemie, Molekulare Genetik, Marie-Curie-Straße 9, D-60439 Frankfurt, Germany;A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Building A’, Moscow 119 899, Russia;Institut für Biochemie, Universitätsklinikum der RWTH Aachen, Pauwelstraße 30, D-52057 Aachen, Germany;Max-Planck-Institut für Biophysik, Abteilung für Molekulare Membranbiologie, Heinrich-Hoffmann-Straße 7, D-60528 Frankfurt, Germany;Johann Wolfgang Goethe-Universität, Institut für Anorganische Chemie und Analytische Chemie, Marie-Curie-Straße 11, D-60439 Frankfurt, Germany

关键词: Heme-copper oxidase;    Heme a propionate;    Non-redox active metal;    Total-reflection X-ray fluorescence spectrometry;    Site-directed mutagenesis;    Structure refinement;    TXRF;    total-reflection X-ray fluorescence spectrometry;    COX;    heme aa 3-type cytochrome c oxidase;    WT;    wild-type;   

DOI  :  10.1016/S0014-5793(99)00977-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown