Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloproteins.

In the present work a data analysis approach, based on XAFS data, is proposed for the identification of most probable binding motifs of unknown mononuclear zinc sites in metalloproteins. This approach combines multiple-scattering EXAFS analysis performed within the rigid-body refinement scheme, nonmuffin- tin ab initio XANES simulations, average structural information on amino acids and metal binding clusters provided by the Protein Data Bank, and Debye–Waller factor calculations based on density functional theory. The efficiency of the method is tested by using three reference zinc proteins for which the local structure around the metal is already known from protein crystallography. To show the applicability of the present analysis to structures not deposited in the Protein Data Bank, the XAFS spectra of six mononuclear zinc binding sites present in diverse membrane proteins, for which we have previously proposed the coordinating amino acids by applying a similar approach, is also reported. By comparing the Zn K-edge XAFS features exhibited by these proteins with those pertaining to the reference structures, key spectral characteristics, related to specific binding motifs, are observed. These case studies exemplify the combined data analysis proposed and further support its validity

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

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

X-ray absorption studies of the local environment of zinc ions bound to the bacterial photosynthetic reaction center

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides slows the light-induced electron and proton transfer to the secondary quinone, QB (Utschig et al., 1998, Biochemistry 37, 8278; Paddock et al., 1999, Proc. Natl. Acad. USA 96, 6183). On the basis of X-ray diffraction (XRD) at 2.5 A ° resolution a site has been identified at the protein surface which binds Cd(II) or Zn(II). Both metal ions binds to the same cluster formed by AspH124, HisH126 and HisH128. A water molecule was also proposed to interact with the Zn (Axelrod et al., 2000, Proc. Natl. Acad. Sci. USA 97, 1542). Recent data suggest that inhibition of proton transfer by Cd(II) is predominantly a consequence of competing with protons for binding to HisH126 and HisH128, thus hampering the function of these residues as proton donor/acceptors along the proton pathway to the QB site (Paddock et al., Biochemistry 42,9626, 2003). Determination of the local structure of the bound metal ions is expected to contribute significantly to elucidate the details of the inhibition mechanism. For this reason we performed Zn K-edge X-ray absorption measurements on Zn-doped RCs embedded into polyvinyl alcohol films at both room and liquid nitrogen temperature. Data analysis has been performed using ab-initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) of significant amplitude have been included. Results for complexes characterized by a Zn to RC stoichiometry close to 1 indicate that Zn binds two O and two N atoms in the first coordination shell. The two N atoms come from His, and only one of the two O atoms comes from an amino acid (Asp or Glu); the second O atom belongs to a water molecule. Complexes characterized by approximately two Zn ions per RC show a second structurally distinct binding site, involving three N and one O atom, all coming from nonaromatic residues. The results obtained for the higher affinity site nicely fit the coordination proposed on the basis of XRD data. The second binding site, revealed by our investigation on noncrystalline samples, is most probably located in a more disordered domain of the RC protein and might have a hitherto not appreciated role in charge transfer inhibition

The Fe2+ Site of Photosynthetic Reaction Centers Probed by Multiple Scattering X-Ray Absorption Fine Structure Spectroscopy: Improving Structure Resolution in Dry Matrices

We report on the x-ray absorption fine structure of the Fe2+ site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe2+ is ligated with four Nɛ atoms from four histidine (His) residues and two Oɛ atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-Nɛ distances, and two distinct Fe-Oɛ bond lengths resolved. The effect of the environment was studied by embedding the reaction centers into a polyvinyl alcohol film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-Nɛ distances, and in one Fe-Oɛ bond length, compared with the polyvinyl alcohol film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the reaction-centers surface and the water-trehalose matrix, which propagates deeply into the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster

EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase

none7noneL. Giachini; F. Francia; F. Boscherini; C. Pacelli; T. Cocco; S. Papa; G. VenturoliL. Giachini; F. Francia; F. Boscherini; C. Pacelli; T. Cocco; S. Papa; G. Venturol

EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase

AbstractThe metal content of bovine NADH-Q oxidoreductase determined by inductively-coupled plasma atomic-emission spectroscopy reveals the presence of about one atom of zinc per molecule of flavin mononucleotide. We applied Zn K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) to investigate the local structure of the bound zinc ion and to identify the nature of the coordinating residues. The EXAFS spectrum is consistent with a structured zinc binding site. By combining information from first-shell analysis and from metalloprotein data bases putative binding clusters have been built and fitted to the experimental spectrum using ab initio simulations. The best fitting binding cluster is formed by two histidine and two cysteine residues arranged in a tetrahedral geometry

X-ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes

Binding of Zn(2+) has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc(1) complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc(1) 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 Zn(2+) bound stoichiometrically to noncrystallized cyt bc(1) 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 His(121), His(268), Lys(270), and Asp(253) residues, and in the homologous bovine enzyme they were the His(121), His(267), Lys(269), and Asp(254) residues. Zn(2+) bound to the bacterial cyt bc(1) 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 His(276), Asp(278), Glu(295), and Asn(279) residues of the cyt b subunit. The conserved location of the Zn(2+) 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