Role of the C-terminal region of Escherichia coli NADH dehydrogenase-2 in membrane anchorage

VILLEGAS, J. M.; VOLENTINI, S. I.; RINTOUL, M. R.; RAPISARDA, V. A.

Congreso; 3rd Latin American Protein Society Meeting and XXXIX Annual Meeting of the Argentinean Biophysical Society; 2010

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane- bound flavoprotein. Preliminary studies suggested that its C-terminal region is involved in membrane anchorage1, 2. We have constructed a water soluble protein, Trun-3, eliminating the last 43 aminoacids of NDH-2. The truncated protein was purified by inmobilized metal affinity chromatography (IMAC) in the absence of detergents throughout the entire process. The activity yield in the purification of Trun-3 from cytosol was ~3-fold higher than the activity recovery of native NDH-2 purified from membrane. Despite the previously described FAD-binding motifs of NDH-2 remain intact in Trun-3, the cofactor was absent in the purified truncated protein. After the addition of 10 ìM FAD, Trun-3 activity was restored and its kinetics constants were measured with different electron acceptors. 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. Our results indicate that NDH-2 C-terminal region is crucial for membrane anchorage. From this region, the amphipathic Arg390-Ala406 helix, predicted by Schmid and Gerloff2, is sufficient to maintain the enzyme bound to the membrane. The mentioned helix contains several aminoacids highly conserved within the known NDH-2s, an unusual feature for a group of proteins that has low percentages of overall sequence identity3. The achievement of the first active and water soluble NDH-2 variant would be useful to solve the protein high-resolution structure and to better understand its catalytic mechanisms.