RESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a classical glycolytic protein that can promote the fusion of phospholipid vesicles and can also play a vital role on in vivo fusogenic events. However, it is not clear how this redox enzyme, which lack conserved structural or sequence motifs related to membrane fusion, catalyze this process. In order to detect if this ability is present in other NAD(P)H dehydrogenases with available structure, spectroscopic studies were performed to evaluate the capability of alcohol dehydrogenase (ADH), glutamic dehydrogenase (GDH) and sorbitol dehydrogenase (SDH) to bind, aggregate, destabilize and fuse vesicles. Based on finite difference Poisson-Boltzmann calculations (FDPB) the protein-membrane interactions were analyzed. A model for the protein-membrane complex in its minimum free energy of interaction was obtained for each protein and the amino acids involved in the binding processes were suggested. A previously undescribed relationship between membrane destabilization and crevices with high electropositive potential on the protein surface was proposed. The putative implication of the non-specific electrostatics on NAD(P)H dehydrogenases induced membrane fusion is discussed.
Assuntos
Fusão de Membrana , NADH NADPH Oxirredutases/química , Lipossomas Unilamelares/química , Álcool Desidrogenase/química , Animais , Sequência de Bases , Bovinos , Sequência Conservada , Desidrogenase de Glutamato (NADP+)/química , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/química , L-Iditol 2-Desidrogenase/química , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilserinas/química , Estrutura Secundária de Proteína , Coelhos , Ovinos , Espectrometria de Fluorescência , Eletricidade Estática , TermodinâmicaRESUMO
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.