FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

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 th...

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Bibliographic Details
Published in:Biochimica et biophysica acta 2014-03, Vol.1844 (3), p.576-584
Main Authors: Villegas, Josefina M., Valle, Lorena, Morán Vieyra, Faustino E., Rintoul, María R., Borsarelli, Claudio D., Rapisarda, Viviana A.
Format: Article
Language:English
Subjects:
Calorimetry, Differential Scanning
Circular Dichroism
Cytosol - enzymology
Escherichia coli
Escherichia coli - enzymology
FAD
Flavin binding
Flavin-Adenine Dinucleotide - metabolism
Flavoprotein
Fluorescence
NADH dehydrogenase
NADH Dehydrogenase - metabolism
Spectrometry, Fluorescence
Spectrophotometry, Ultraviolet
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Summary: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)×104M−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. •Conformational state of truncated NDH-2 is not strongly influenced by FAD binding.•FAD binding does not modify thermal stability of truncated NDH-2.•FAD–protein binding occurred with a 1:1 stoichiometry and a KA=7.0×104M−1.
ISSN:1570-9639
0006-3002
1878-1454
1878-2434
DOI:10.1016/j.bbapap.2013.12.021