Cytochrome c oxidase (COX) is the terminal component of the respiratory chain: it catalyses the oxidation of cytochrome c reduced by the cytochrome bc1 complex, reducing O2 to H2O and pumping protons across the mitochondrial membrane. Definition of the proton transfer pathways is an open question, crucial for understanding the catalytic mechanism of the enzyme (1). Zn2+ inhibits bovine heart COX (2). The observation that Zn2+ inhibits proton uptake in the bacterial cytochrome c oxidase led to propose that Zn2+ binds near the H+ entry point of the Dpathway where a cluster of histidine residues and carboxylates is found (3). An endogenous Zn ion has been found in the bovine heart COX where it binds to a structurally well defined site (4) . To characterize the site at which exogenous Zn binds, inhibiting proton uptake, we performed Zn K-edge X-ray Absorption Spectroscopy (XAS) measurements on three samples of bovine COX characterized respectively by 1, 1.5 and 2 zinc atoms per complex. The last two samples were obtained by incubating the protein with a proper amount of ZnSO4. Metal stoichiometries were measured by ICP-emission spectroscopy. In agreement with X-ray diffraction data (4), XAS analysis of the endogenous zinc site shows that Zn binds four sulphur atoms. The average Zn-S distance is slightly bigger (2.33 Å) than that reported in the crystallographic model (2.25 Å). On the basis of the XAS spectrum of the endogenous Zn, we extracted the XAS signal of the inhibitory Zn binding site(s). The difference spectra obtained from samples characterized by 1.5 and 2.0 Zn per complex exhibited very similar features. This indicates the presence of a unique, high affinity zinc binding site. First shell analysis of the XAS signal for the exogenous Zn site suggests three nitrogen atoms at 1.99 Å and one O at 1.98 Å as ligands. A complete, multiple-shell, multiple scattering analysis is in progress to possibly localize a cluster of residues consistent with the XAS local structure.
Francia F., Giachini L., Capitanio G., Martino L., Papa S., Boscherini F., et al. (2006). The local structure of Zn binding sites in the bovine cytochrome c oxidase: An X-ray Absorption Spectroscopy study. Elsevier.
The local structure of Zn binding sites in the bovine cytochrome c oxidase: An X-ray Absorption Spectroscopy study
FRANCIA, FRANCESCO;GIACHINI, LISA;BOSCHERINI, FEDERICO;VENTUROLI, GIOVANNI
2006
Abstract
Cytochrome c oxidase (COX) is the terminal component of the respiratory chain: it catalyses the oxidation of cytochrome c reduced by the cytochrome bc1 complex, reducing O2 to H2O and pumping protons across the mitochondrial membrane. Definition of the proton transfer pathways is an open question, crucial for understanding the catalytic mechanism of the enzyme (1). Zn2+ inhibits bovine heart COX (2). The observation that Zn2+ inhibits proton uptake in the bacterial cytochrome c oxidase led to propose that Zn2+ binds near the H+ entry point of the Dpathway where a cluster of histidine residues and carboxylates is found (3). An endogenous Zn ion has been found in the bovine heart COX where it binds to a structurally well defined site (4) . To characterize the site at which exogenous Zn binds, inhibiting proton uptake, we performed Zn K-edge X-ray Absorption Spectroscopy (XAS) measurements on three samples of bovine COX characterized respectively by 1, 1.5 and 2 zinc atoms per complex. The last two samples were obtained by incubating the protein with a proper amount of ZnSO4. Metal stoichiometries were measured by ICP-emission spectroscopy. In agreement with X-ray diffraction data (4), XAS analysis of the endogenous zinc site shows that Zn binds four sulphur atoms. The average Zn-S distance is slightly bigger (2.33 Å) than that reported in the crystallographic model (2.25 Å). On the basis of the XAS spectrum of the endogenous Zn, we extracted the XAS signal of the inhibitory Zn binding site(s). The difference spectra obtained from samples characterized by 1.5 and 2.0 Zn per complex exhibited very similar features. This indicates the presence of a unique, high affinity zinc binding site. First shell analysis of the XAS signal for the exogenous Zn site suggests three nitrogen atoms at 1.99 Å and one O at 1.98 Å as ligands. A complete, multiple-shell, multiple scattering analysis is in progress to possibly localize a cluster of residues consistent with the XAS local structure.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
