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Inhibition of pea ferredoxin–NADP(H) reductase by Zn‐ferrocyanide

2004; Wiley; Volume: 271; Issue: 22 Linguagem: Inglês

10.1111/j.1432-1033.2004.04430.x

1432-1033

Daniela L. Catalano‐Dupuy, Daniela V. Rial, Eduardo A. Ceccarelli,

Metal-Catalyzed Oxygenation Mechanisms

Ferredoxin–NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn 2+ ( K i 1.57 µ m ). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn 2+ ( K i 1.80 µ m ), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions. Escherichia coli FNR was also inhibited by Zn‐ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron‐transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn‐ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn‐ferrocyanide in a noncompetitive manner, but the observed K i was about nine times higher than those for the diaphorase reactions. The electron transfer to Anabaena flavodoxin was not affected by Zn‐ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn‐ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect.