Calcium-Binding Sites of Calmodulin and Electron Transfer by Inducible Nitric Oxide Synthase

• Published in: Biochemistry (Easton) 2005-05, Vol.44 (20), p.7593-7601
• Main Authors: Gribovskaja, Irena, Brownlow, Kaleb C, Dennis, Sam J, Rosko, Andrew J, Marletta, Michael A, Stevens-Truss, Regina
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0474517

Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca2+-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca2+-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 Ca2+-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 Ca2+-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca2+ 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 Ca2+ being bound to all four Ca2+-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.