title: Inhibition of pea ferredoxin-NADP(H) reductase by Zn-ferrocyanide.
abstract: 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 microM). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 microM), 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 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.
authors.affiliation: Molecular Biology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
authors.forename: Daniela L Catalano
authors.initials: DL
authors.lastname: Dupuy
authors.forename: Daniela V
authors.initials: DV
authors.lastname: Rial
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Ceccarelli
mesh_terms: D015086:2,6-Dichloroindophenol / Q000737:chemistry / Q000494:pharmacology; D019943:Amino Acid Substitution; D001665:Binding Sites; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005287:Ferredoxin-NADP Reductase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005295:Ferrocyanides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009536:Niacinamide / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology*; D010940:Plant Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D005295:Ferrocyanides; D005415:Flavins; D005418:Flavodoxin; D010940:Plant Proteins; D011994:Recombinant Proteins; D009536:Niacinamide; D009249:NADP; D015086:2,6-Dichloroindophenol; D005287:Ferredoxin-NADP Reductase; D015032:Zinc