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Transhydrogenase couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. The binding of Zn(2+) to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn(2+) bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which betaHis91 was replaced by Lys (betaH91K). This well-conserved His residue, located in the membrane-spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn(2+). The XAFS analysis has identified a Zn(2+)-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn(2+) inhibits proton translocation by competing with H(+) binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and betaH91K. Evidently, betaHis91 is not directly involved in Zn(2+) binding. The locus of betaHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.
Pubmed ID: 20080075
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Athena is a web-based application that warehouses disparate datatypes related to the control of gene expression. Athena provides several features to enable exploration of the regulatory mechanisms of Arabidopsis gene control. The first main tool we provide is visualization of promoter domains of selected genes. Database crossreference for these transcription factors is provided as well as a statistical test for enrichment of binding activity within the set of selected promoters. The data mining tools in Athena allow for selection of sets of genes based on two different factors. -Genes can be select by specifying a set of binding factors whose putative sites must be present within all of those genes'' promoter regions. -Alternatively, genes can be selected using Gene Ontology annotations. Both GO (Gene Ontology) Slim terms and Gene Ontology terms are available. One can select a set of genes by either choosing a union of the genes annotated by a selected set of Slim terms or Gene Ontology terms. The selected gene''s putative binding factors are listed, including enrichment data. Furthermore, enriched presence of Gene Ontology terms is given. The analysis suite provides both enhanced data mining tools for selecting genes as well as several data displays.
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