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X-Ray Absorption Studies of Zn²⁺ Binding Sites in Bacterial, Avian, and Bovine Cytochrome bc₁ Complexes

Lisa Giachini ^* , Francesco Francia , Giulia Veronesi ^*, Dong-Woo Lee , Fevzi Daldal , Li-Shar Huang , Edward A. Berry , Tiziana Cocco ^¶, Sergio Papa ^¶, Federico Boscherini ^* || and Giovanni Venturoli  ^||

^* Department of Physics and Department of Biology, University of Bologna, Bologna, Italy; Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania; Lawrence Berkeley National Laboratory, Berkeley, California; ^¶ Department of Medical Biochemistry, Biology and Physics, University of Bari, Bari, Italy; and ^|| Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, Bologna, Italy

Correspondence: Address reprint requests to Giovanni Venturoli, Dept. of Biology, University of Bologna, Bologna, Italy. E-mail: ventur{at}alma.unibo.it.

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 Q_o site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Q_o 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.

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