Modulation of the remote heme site geometry of recombinant mouse neuronal nitric-oxide synthase by the N-terminal hook region

J Biol Chem. 1999 Mar 19;274(12):7705-13. doi: 10.1074/jbc.274.12.7705.


The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated. Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems. The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wild-type enzyme. Replacement of Asp-314 by Ala altered the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogated the ability of nNOS to form catalytically active dimers. Replacement of Thr-315 by Ala reduced the protein stability and altered the geometry around the heme site, especially in the absence of bound (6R)-5,6,7, 8-tetrahydro-L-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Catalytic Domain
  • Cattle
  • Crystallography, X-Ray
  • Dimerization
  • Drosophila
  • Electron Spin Resonance Spectroscopy
  • Heme / chemistry*
  • Humans
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nitric Oxide Synthase / chemistry*
  • Nitric Oxide Synthase Type I
  • Protein Conformation
  • Protein Folding
  • Rats
  • Recombinant Proteins / chemistry
  • Sequence Alignment
  • Structure-Activity Relationship


  • Recombinant Proteins
  • Heme
  • NOS1 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type I
  • Nos1 protein, mouse
  • Nos1 protein, rat