EFFECT OF THE MUTATIONS ON THE Cys RESIDUES IN THE Escherichia coli NADH-DEHYDROGENASE-2 ACTIVITIES

VOLENTINI SI; SOLBIATI J; RAPISARDA VA; RODRIGUEZ MONTELONGO L; FARIAS RN

Bariloche

Congreso; Reunion anual de SAIB; 2003

SAIB

NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. Our previous studies showed that purified NDH-2 has a Cu(I)-bound and exhibits Cu(II)-reductase activity supported by NADH. Moreover, bioinformatic analysis indicated that this protein has a cysteine-rich domain (domain III) conserved in primary and secondary structure with the HMA (Heavy Metal Associated) domains that could be the site of copper binding. Domain III presents the Cys315XXCys318 and the MetXXCys339 motifs. In order to study the participation of all the Cys present in domain III on the NDH-2 activities, we prepared a set of plasmids bearing ndh gene mutagenized in the three Cys and then transformed into strain IY12 that lacks the NADH- dehydrogenase activities (nuo and ndh). We found that the mutations affected the NADH-dehydrogenase and the NADH-oxidase activities of the membranes, compared with one preparation containing the wild-type protein. We saw that both activities decreased about 80 % in the C318S mutant, and almost disappeared in the double mutant (C315S-C318S). On other hand, the activities increased about 50 % in C339S mutants. These results indicating that this mutations affected some of the NDH-2 activities, suggest that Cys315XXCys318 and MetXXCys339 motifs are involved on the functionality of the enzyme directly or due to the Cu (I) putatively bound there.