RAPISARDA Viviana Andrea
congresos y reuniones científicas
The role of Cys318 in the enzymatic activities of the Cu(II)-reductase NADH dehydrogenase-2
VOLENTINI, S. I.; SOLBIATI, J. O.; FARÍAS, R. N.; RODRÍGUEZ-MONTELONGO, L.; RAPISARDA, V. A.
Iguazú, Misiones (Argentina)
Congreso; XL Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2004
NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane flavoprotein coded by ndh gene. We described previously that it is Cu(II)-reductase and it has a Cu(I) atom bound per polypeptidic chain. The protein contains 4 cysteines: Cys315, Cys318, and Cys339 in a Heavy-Metal-Associated Motif, and Cys137 in a predicted flexible loop. To study the relevance of the Cys residues in NDH-2 we constructed single and combined-double mutants of the protein, changing Cys by Ser. We assayed the dehydrogenase and cupric-reductase activities of the enzyme either on membranes or on detergent solubilized extracts. Results demonstrated that the substitution of the Cys318 led to a 50-70% decrease of the activities compared with the wild-type protein. Also the double mutants C315S-C318S and C137S-C318S diminished the activities in comparable percentages to the simple mutant. These mutants presenting low activities are deficient to grow on mannitol as it is the ndh- strain. On the other hand, the mutant C339S-C318S showed an increased of 20-50% in the activities respect to the wild-type and recovers the capacity to grow on mannitol. Previous results of the group demonstrated an increase in all the activities, for the single mutant C339S. These results suggest that Cys318 residue in NDH-2 would be directly involved in the protein activities and/or the copper binding. We discuss the possible participation of Cys339 as redox-regulator.