Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase

Biochemistry. 2005 May 24;44(20):7593-601. doi: 10.1021/bi0474517.


Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / genetics
  • Calcium / chemistry*
  • Calcium / metabolism
  • Calmodulin / chemistry*
  • Calmodulin / metabolism
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism
  • Electron Transport / genetics
  • Enzyme Activation / genetics
  • Holoenzymes / biosynthesis
  • Holoenzymes / chemistry
  • Holoenzymes / genetics
  • Holoenzymes / isolation & purification
  • Nerve Tissue Proteins / metabolism
  • Nitric Oxide Synthase / biosynthesis
  • Nitric Oxide Synthase / chemistry*
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase / isolation & purification
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type I
  • Nitric Oxide Synthase Type II
  • Protein Binding / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification


  • Calmodulin
  • Drosophila Proteins
  • Holoenzymes
  • Nerve Tissue Proteins
  • Recombinant Proteins
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type I
  • Nitric Oxide Synthase Type II
  • Calcium