Inhibición por Zn2+ de la Ferredoxina-NADP+ oxidoreductasa de arveja

CATALANO DUPUY, DANIELA L.; RIAL, DANIELA V; CECCARELLI, EDUARDO A

Villa Carlos Paz, Córdoba, Argentina

Congreso; XXXVIII Reunión Anual de SAIB; 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Ferredoxin-NADP(H) reductases (FNR) constitute a family of hydrophilic, monomeric enzymes that contain noncovanlently bound FAD. In chloroplasts, they catalyze the final 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 artificial 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 in a pH dependent manner. The estimated value of Ki is in the low micromolar range. Moreover, this inhibition is reversed instantaneously and completely by adding 1mM EDTA.  However, using DCPIP as electron acceptor, diaphorase activity is only inhibited in the presence of equimolar concentrations of ferrocyanide and zinc. Likewise, this condition is necessary for inhibiting Fd reduction, suggesting that the complex of ferrocyanide and zinc is the true inhibitor. Finally, we found that NADPH can efficiently reduce the flavoprotein in the presence of an inhibitory concentration of ferrocyanide-zinc, indicating that the observed inhibition is not at this level.  We propose that there is a ferrocyanide-zinc binding site in the FNR and it probably involves the isoalloxazine moiety of FAD.