Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase.
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.
Gribovskaja
Irena
I
Department of Chemistry, Kalamazoo College, Kalamazoo, Michigan 49006, USA.
Brownlow
Kaleb C
KC
Dennis
Sam J
SJ
Rosko
Andrew J
AJ
Marletta
Michael A
MA
Stevens-Truss
Regina
R
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D020035:Holoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D002147:Calmodulin; D029721:Drosophila Proteins; D020035:Holoenzymes; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D002118:Calcium