A Zn-ferrocyanide complex inhibits Ferredoxin-NADP+ oxidoreductase activities

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

Bariloche, Río Negro, ARGENTINA

Congreso; XXXIX Reunión Anual de SAIB; 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Ferredoxin-NADP(H) reductases (FNR) are hydrophilic, monomeric enzymes that contain FAD. They catalyze the final step of photosynthetic electron transport from ferredoxin (Fd) to NADP+ generating NADPH for biosynthetic pathways. NADPH oxidation can be assayed using ferricyanide, 2,6-dichlorophenol indophenol (DCPIP), other substituted phenols as artificial electron acceptors (diaphorase), or the natural electron transport protein Fd. In addition to its catalytic, co-catalytic, and/or structural roles in proteins, zinc inhibits several enzymes. We found that FNR ferricyanide-diaphorase activity is inhibited by zinc. Equimolar concentrations of the metal and ferrocyanide are necessary for inhibiting DCPIP-diaphorase activity and Fd reduction, suggesting that the complex is the true inhibitor. Kinetics studies showed that Zn-ferrocyanide behaves as a non-competitive inhibitor in all FNR enzymatic activities. Moreover, the dissociating constants (KD) of the FNR-Fd and FNR-NADP+ complexes do not change with inhibiting concentrations of zinc-ferrocyanide. Inactivation experiments vs time reflected that the diaphorase activity is considerably reduced in the presence of the inhibitor, probably due to the loss of the prosthetic group FAD. Finally, we found that FNR from E. coli (FPR) is inhibited by zinc-ferrocyanide similarly to the pea FNR. Our results depict a structural particularity likely to be present in most of the flavoenzymes, which reveals structure-function information about them.