title: FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2.
abstract: Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.
authors.affiliation: Instituto de Química Biológica "Dr Bernabé Bloj", Facultad de Bioquímica, Química y Farmacia (UNT) and Instituto Superior de Investigaciones Biológicas, INSIBIO, CCT-Tucumán (CONICET-UNT), Chacabuco 461, T4000ILI Tucumán, Argentina.
authors.forename: Josefina M
authors.initials: JM
authors.lastname: Villegas
authors.affiliation: Laboratorio de Cinética y Fotoquímica, Centro de Investigaciones y Transferencia de Santiago del Estero (CITSE-CONICET), Universidad Nacional de Santiago del Estero (UNSE), RN9 Km 1125, Villa el Zanjón, 4206 Santiago del Estero, Argentina.
authors.forename: Lorena
authors.initials: L
authors.lastname: Valle
authors.affiliation: Laboratorio de Cinética y Fotoquímica, Centro de Investigaciones y Transferencia de Santiago del Estero (CITSE-CONICET), Universidad Nacional de Santiago del Estero (UNSE), RN9 Km 1125, Villa el Zanjón, 4206 Santiago del Estero, Argentina.
authors.forename: Faustino E
authors.initials: FE
authors.lastname: Morán Vieyra
authors.affiliation: Instituto de Química Biológica "Dr Bernabé Bloj", Facultad de Bioquímica, Química y Farmacia (UNT) and Instituto Superior de Investigaciones Biológicas, INSIBIO, CCT-Tucumán (CONICET-UNT), Chacabuco 461, T4000ILI Tucumán, Argentina.
authors.forename: María R
authors.initials: MR
authors.lastname: Rintoul
authors.affiliation: Laboratorio de Cinética y Fotoquímica, Centro de Investigaciones y Transferencia de Santiago del Estero (CITSE-CONICET), Universidad Nacional de Santiago del Estero (UNSE), RN9 Km 1125, Villa el Zanjón, 4206 Santiago del Estero, Argentina. Electronic address: cdborsarelli@gmail.com.
authors.forename: Claudio D
authors.initials: CD
authors.lastname: Borsarelli
authors.affiliation: Instituto de Química Biológica "Dr Bernabé Bloj", Facultad de Bioquímica, Química y Farmacia (UNT) and Instituto Superior de Investigaciones Biológicas, INSIBIO, CCT-Tucumán (CONICET-UNT), Chacabuco 461, T4000ILI Tucumán, Argentina. Electronic address: vrapisarda@fbqf.unt.edu.ar.
authors.forename: Viviana A
authors.initials: VA
authors.lastname: Rapisarda
mesh_terms: D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D009245:NADH Dehydrogenase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005182:Flavin-Adenine Dinucleotide; D009245:NADH Dehydrogenase
keywords: FAD; Flavin binding; Flavoprotein; Fluorescence; NADH dehydrogenase