X-Ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes.
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, we report here on the local structure of Zn2+ bound stoichiometrically to noncrystallized cyt bc1 complexes. We performed a comparative x-ray absorption fine-structure spectroscopy study by examining avian, bovine, and bacterial enzymes. A large number of putative clusters, built by combining information from first-shell analysis 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 His121, His267, Lys269, and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of 6. The best-fit octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue, and two water molecules. It was interesting that by aligning the crystallographic structures of the bacterial and avian enzymes, 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 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 donors/acceptors.
Giachini
Lisa
L
Department of Physics, University of Bologna, Bologna, Italy.
Francia
Francesco
F
Veronesi
Giulia
G
Lee
Dong-Woo
DW
Daldal
Fevzi
F
Huang
Li-Shar
LS
Berry
Edward A
EA
Cocco
Tiziana
T
Papa
Sergio
S
Boscherini
Federico
F
Venturoli
Giovanni
G
D000818:Animals; D001665:Binding Sites; D001717:Birds; D002417:Cattle; D003198:Computer Simulation; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013045:Species Specificity; D015032:Zinc / Q000737:chemistry*
D014450:Electron Transport Complex III; D015032:Zinc