Molecular architecture of mammalian nitric oxide synthases

Proc Natl Acad Sci U S A. 2014 Sep 2;111(35):E3614-23. doi: 10.1073/pnas.1413763111. Epub 2014 Aug 14.


NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms--inducible, endothelial, and neuronal NOS--that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.

Keywords: conformational heterogeneity; electron microscopy; flavin; heme.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calmodulin / chemistry
  • Calmodulin / metabolism
  • Crystallization
  • Dimerization
  • Holoenzymes / chemistry
  • Holoenzymes / metabolism
  • Humans
  • Imaging, Three-Dimensional
  • Mammals
  • Mice
  • Microscopy, Electron, Transmission
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type I / chemistry*
  • Nitric Oxide Synthase Type I / metabolism
  • Nitric Oxide Synthase Type II / chemistry*
  • Nitric Oxide Synthase Type II / metabolism
  • Nitric Oxide Synthase Type III / chemistry*
  • Nitric Oxide Synthase Type III / metabolism
  • Oxidoreductases / chemistry
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Rats


  • Calmodulin
  • Holoenzymes
  • Nitric Oxide
  • Oxidoreductases
  • Nitric Oxide Synthase Type I
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase Type III
  • Nos1 protein, mouse
  • Nos2 protein, mouse
  • Nos3 protein, mouse