Inhibition of pea ferredoxin-NADP+ reductase by Zn-ferrocyanide.

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

EUROPEAN JOURNAL OF BIOCHEMISTRY

Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies

Lugar: Cambridge, UK; Año: 2004 vol. 271 p. 4582 - 4593

0014-2956

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 Zn2+ (Ki 1.57 mM). Dichlorophenolindophenol daphorase activity was also inhibited by Zn2+ (Ki 1.80 mM), 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 Ki 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 over-come the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners that alters or completely prevents the inhibitory effect.