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

Abstract

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca 2+-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca 2+ with mutations in the N-lobe (CaM 12), the C-lobe (CaM 34), or both lobes of CaM (CaM 1234) to determine their effect on the binding and activation of the Ca 2+-dependent neuronal (nNOS) and Ca 2+-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 Ca 2+. Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.