Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca2+-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca2+ with mutations in the N-lobe (CaM12), the C-lobe (CaM34), or both lobes of CaM (CaM1234) to determine their effect on the binding and activation of the Ca2+-dependent neuronal (nNOS) and Ca2+-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein–protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca2+. Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.