RAPISARDA Viviana Andrea
congresos y reuniones científicas
Changes induced by FAD binding on a truncated NADH dehydrogenase-2
VILLEGAS, J. M.; RINTOUL, M. R.; RAPISARDA, V. A.
Workshop; WABFQP, 1er Workshop Argentino de Biofísicoquímica de Proteínas. Taller satélite del XVII congreso de Fisicoquímica y Química Inorgánica; 2011
Introduction: Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein. Preliminary reports have predicted how NDH-2 is attached to the membrane using only bioinformatics approaches, but this issue has been controversial (1, 2). By constructing a soluble NDH-2 variant (Trun-3), lacking the last 43 aminoacids, we have concluded that C-terminal region is crucial for the protein membrane anchorage. Trun-3 was then purified by inmobilized metal affinity chromatography (IMAC) in the absence of detergents throughout the entire process. Despite the putative FAD-binding motifs of NDH-2 remain intact in Trun-3, this enzyme only showed activity after the addition of 10 µM FAD. Aim: To determine kinetics and structural properties of the water soluble NDH-2 variant. Results: Trun-3 kinetics constants were measured with different electron acceptors upon the addition of FAD. It was observed that Trun-3 maximum velocity was similar to NDH-2 activity with ferrycianide and MTT, while it was 5-fold lower with quinones. However, no significant difference was observed in Km values for both enzymes with any of the tested acceptors. By fluorescence analysis, we verified the absence of FAD cofactor in Trun-3. To examine the conformational changes induced in the truncated protein by its cofactor binding, a limited digestion experiment with trypsin was performed. Denaturing polyacrylamide gel electrophoresis revealed a different fragmentation pattern in the presence of FAD compared to that of the apo-Trun-3. Conclusions: We performed a kinetics characterization of Trun-3 using as electron acceptors several ubiquinone analogous as well as artificial acceptors. These results suggest that C-terminal region could play key roles maintaining both the FAD bound to the polypeptide and the correct quinone orientation during the catalysis. On the other hand, the protease digestion assay revealed that FAD binding has a widespread effect on the structure of Trun-3. Apparently, FAD is able to induce a stable conformation in Trun-3.