title: Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase.
abstract: 14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.
authors.affiliation: Institute of Biochemistry, Department of Chemistry and Center for Molecular Medicine, University of Cologne, 50674 Cologne, Germany.
authors.forename: Iris C
authors.initials: IC
authors.lastname: Lambeck
authors.forename: Katrin
authors.initials: K
authors.lastname: Fischer-Schrader
authors.forename: Dimitri
authors.initials: D
authors.lastname: Niks
authors.forename: Juliane
authors.initials: J
authors.lastname: Roeper
authors.forename: Jen-Chih
authors.initials: JC
authors.lastname: Chi
authors.forename: Russ
authors.initials: R
authors.lastname: Hille
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology; D006418:Heme / Q000235:genetics / Q000378:metabolism; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary
chemical_list: D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D006418:Heme; D050901:Nitrate Reductase