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

J.M. VILLEGAS; L. VALLE; F. E. MORÁN VIEYRA; M. R. RINTOUL; C. D. BORSARELLI; V. A. RAPISARDA

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2014 vol. 1844 p. 576 - 584

1570-9639

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 be- tween 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 emis- sion of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp res- idues. 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)× 104 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.