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

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

EUROPEAN JOURNAL OF BIOCHEMISTRY

Blackwell - Synergy

Lugar: Londres; Año: 2004 vol. 271 p. 4582 - 4593

0014-2956

Ferredoxin–NADP(H) reductases (FNRs) represent a prototypeof enzymes involved in numerous metabolic pathways.We found that pea FNR ferricyanide diaphoraseactivity was inhibited by Zn2+ (Ki 1.57 lM). Dichlorophenolindophenoldiaphorase activity was also inhibited byZn2+ (Ki 1.80 lM), but the addition of ferrocyanide wasrequired, indicating that the inhibitor is an arrangement ofboth ions. Escherichia coli FNR was also inhibited byZn-ferrocyanide, suggesting that inhibition is a consequenceof common structural features of these flavoenzymes. Theinhibitor behaves in a noncompetitive manner for NADPHand for artificial electron acceptors. Analysis of the oxidationstate of the flavin during catalysis in the presence of theinhibitor suggests that the electron-transfer process betweenNADPH and the flavin is not significantly altered, and thatthe transfer between the flavin and the second substrate ismainly affected. Zn-ferrocyanide interacts with the reductase,probably increasing the accessibility of the prostheticgroup to the solvent. Ferredoxin reduction was also inhibitedby Zn-ferrocyanide in a noncompetitive manner, but theobserved Ki was about nine times higher than those for thediaphorase reactions. The electron transfer to Anabaenaflavodoxin was not affected by Zn-ferrocyanide. Binding ofthe apoflavodoxin to the reductase was sufficient to overcomethe inhibition by Zn-ferrocyanide, suggesting that theinteraction of FNRs with their proteinaceous electronpartners may induce a conformational change in thereductase that alters or completely prevents the inhibitoryeffect.