Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites, located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge X-ray absorption fine structure spectroscopy (XAFS) we report here on the local structure of Zn2+ bound stoichiometrically to non crystallized cyt bc1 complexes. We performed a comparative XAFS study by examining the avian, the bovine and the bacterial enzymes. A large number of putative clusters, built by combining information from firstshell nalysis and metalloprotein databases, were fitted to the experimental spectra by using ab initio simulations. This procedure led us to identify the binding clusters with high levels of confidence. In both the avian and bovine enzyme a tetrahedral ligand cluster formed by two His, one Lys and one carboxylic residue was found, and this ligand attribution fit the crystallographic Zn01 location of the avian enzyme. In the chicken enzyme the ligands were the His121, His268, Lys270 and Asp253 residues, and in the homologous bovine enzyme they were the His 121, His267, Lys269 and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of six. The best fitting octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue and two water molecules. Interestingly, by aligning the crystallographic structures of the bacterial and avian enzyme, this group of residues was found located in the region homologous to that of the Zn01 site. This cluster included the His276, Asp278, Glu295 and Asn279 residues of the cyt b subunit. The conserved location of the Zn2+ binding sites at the entrance of the putative proton release pathways, and the presence of His residues point out to a common mechanism of inhibition. As previously shown for the photosynthetic bacterial reaction center, zinc would compete with protons for binding to the His residues, thus impairing their function as proton donor/acceptors.

Comparative study of the binding sites of Zn: a proton transfer inhibitor in respiratory enzymes. SC-1853.

VENTUROLI, GIOVANNI;FRANCIA, FRANCESCO;BOSCHERINI, FEDERICO;GIACHINI, LISA;
2005

Abstract

Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites, located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge X-ray absorption fine structure spectroscopy (XAFS) we report here on the local structure of Zn2+ bound stoichiometrically to non crystallized cyt bc1 complexes. We performed a comparative XAFS study by examining the avian, the bovine and the bacterial enzymes. A large number of putative clusters, built by combining information from firstshell nalysis and metalloprotein databases, were fitted to the experimental spectra by using ab initio simulations. This procedure led us to identify the binding clusters with high levels of confidence. In both the avian and bovine enzyme a tetrahedral ligand cluster formed by two His, one Lys and one carboxylic residue was found, and this ligand attribution fit the crystallographic Zn01 location of the avian enzyme. In the chicken enzyme the ligands were the His121, His268, Lys270 and Asp253 residues, and in the homologous bovine enzyme they were the His 121, His267, Lys269 and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of six. The best fitting octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue and two water molecules. Interestingly, by aligning the crystallographic structures of the bacterial and avian enzyme, this group of residues was found located in the region homologous to that of the Zn01 site. This cluster included the His276, Asp278, Glu295 and Asn279 residues of the cyt b subunit. The conserved location of the Zn2+ binding sites at the entrance of the putative proton release pathways, and the presence of His residues point out to a common mechanism of inhibition. As previously shown for the photosynthetic bacterial reaction center, zinc would compete with protons for binding to the His residues, thus impairing their function as proton donor/acceptors.
2005
G. Venturoli; F. Francia; F. Boscherini; L. Giachini; Fevzi Daldal; Sergio Papa
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/151662
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