title: Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2.
abstract: NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress. In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties. Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm. This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA. The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein. There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy. Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus. A NDH-2 topology model, based on the secondary structure prediction, is proposed. This is the first description of a copper-containing NADH dehydrogenase. Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.
authors.affiliation: Departamento Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas, Universidad Nacional de Tucumán, Chacabuco 461, San Miguel de Tucumán, Argentina.
authors.forename: Viviana A
authors.initials: VA
authors.lastname: Rapisarda
authors.forename: Rosana N
authors.initials: RN
authors.lastname: Chehín
authors.forename: Javier
authors.initials: J
authors.lastname: De Las Rivas
authors.forename: Luisa
authors.initials: L
authors.lastname: Rodríguez-Montelongo
authors.forename: Ricardo N
authors.initials: RN
authors.lastname: Farías
authors.forename: Eddy M
authors.initials: EM
authors.lastname: Massa
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism*; D018384:Oxidative Stress; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D009942:Organometallic Compounds; C048402:copper(I)-thiolate; D003300:Copper; D009245:NADH Dehydrogenase