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

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‐dir...

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Bibliographic Details
Published in:FEBS letters 1999-08, Vol.456 (3), p.365-369
Main Authors: Pfitzner, Ute, Kirichenko, Anna, Konstantinov, Alexander A., Mertens, Martina, Wittershagen, Axel, Kolbesen, Bernd O., Steffens, Guy C.M., Harrenga, Axel, Michel, Hartmut, Ludwig, Bernd
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Calcium - metabolism
Cattle
Copper - metabolism
COX, heme aa 3-type cytochrome c oxidase
Crystallography, X-Ray
Electron Transport Complex IV - chemistry
Electron Transport Complex IV - genetics
Electron Transport Complex IV - metabolism
Heme a propionate
Heme-copper oxidase
Iron - metabolism
Mitochondria - enzymology
Mutation
Non-redox active metal
Paracoccus denitrificans - enzymology
Protein Conformation
Site-directed mutagenesis
Sodium - chemistry
Sodium - metabolism
Spectrometry, X-Ray Emission
Structure refinement
Total-reflection X-ray fluorescence spectrometry
TXRF, total-reflection X-ray fluorescence spectrometry
Water
WT, wild-type
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Summary: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.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(99)00977-1