Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

J Biol Chem. 2012 Feb 10;287(7):4562-71. doi: 10.1074/jbc.M111.323113. Epub 2011 Dec 13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism*
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Electron Transport / physiology
  • Heme / genetics
  • Heme / metabolism
  • Nitrate Reductase / genetics
  • Nitrate Reductase / metabolism*
  • Oxidation-Reduction
  • Phosphorylation / physiology
  • Protein Binding / physiology
  • Protein Multimerization / physiology*
  • Protein Structure, Tertiary

Substances

  • 14-3-3 Proteins
  • Arabidopsis Proteins
  • Heme
  • Nitrate Reductase