Presentación: Differential effects of Zn-ferrocyanide on pea ferredoxin-NADP+ reductase activities

CATALANO DUPUY, D; RIAL, D; CECCARELLI, EDUARDO A.

Río de Janeiro

Congreso; First Latin-American Protein Society - Meeting, Angra dos Reis; 2004

Latinamerican Protein Society

Ferredoxin-NADP(H) reductases (FNR) constitute a family of hydrophilic, monomeric enzymes that contain noncovanlently bound FAD. In chloroplasts, they catalyze the _nal step of photosynthetic electron transport from ferredoxin (Fd) to NADP+ with formation of the NADPH necessary for biosynthetic pathways. Diaphorase activity, catalyzing NADPH oxidation, can be assayed using ferricyanide, 2,6-dichlorophenol indophenol (DCPIP) or other substitutes phenols as arti_cial electron acceptors. The electron transport from NADPH to Fd can also be measured in vitro. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of proteins, zinc inhibits several enzymes. We found that FNR diaphorase activity, using ferricyanide as electron acceptor, is inhibited by zinc ion. Diaphorase activity, using DCPIP as electron acceptor is also inhibited but equimolar concentrations of the metal and ferrocyanide are necessary for the e_ect, suggesting that an arrangement of ferrocyanide and zinc is the true inhibitor. Kinetics studies of these activities in the presence of di_erent concentrations of the inhibitor show that it behave in a non-competitive manner. Moreover, the dissociation constants (KD) of the FNR-Fd and FNR-NADP+ complexes are not a_ected by Zn-ferrocyanide. Fluorescence measurements indicate that Zn-ferrocyanide interacts directly with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Fd reduction is also inhibited by Zn-ferrocyanide but the observed Ki is 8 times higher than the one for the diaphorase reaction. In addition, the electron transfer to Anabaena avodoxin is not a_ected by Zn-ferrocyanide and the binding of the apoavodoxin to the reductase was su_cient to overcome the inhibition produced by Zn-ferrocyanide on the diaphorase activity. We suggest that the interaction of FNR with their proteinaceus electron partners induces a noticeable conformational change in the reductase, and that it is more signi_cant when avodoxin is bound. Finally, we found that the Ferredoxin-NADP+ oxidoreductase of E. coli (Fpr) is also inhibited by Zn-ferrocyanide, suggesting the inhibition could be the result of a structural particularity of all avoenzymes.