The 42-Amino Acid Insert in the FMN Domain of Neuronal Nitric-oxide Synthase Exerts Control over Ca2+/Calmodulin-dependent Electron Transfer

Abstract

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

“…Peptides corresponding to caveolin-1 residues 82-101 (DGIWKASFTTFTVTKY-WFYR: termed CaV1p1), 135-156 (DLPFEAIGKISNIRINTQKEI), and caveolin-3 residues 109 -130 (KSYLIEIQCISHIYSLCIRTFC) and residues 65-84 (DGVWRVSYTTFTVSKYWCYR) were synthesized by Sawady Technology (Tokyo, Japan) and purified by high pressure liquid chromatography to a purity of Ͼ98% as confirmed by mass spectrometric analysis. The control peptides (PPG) 5 and apamin (CNCKAPETAL-CARRCQQH) were purchased from Peptide Laboratory (Osaka, Japan (16), the C-terminal truncation mutant (⌬C33, a deletion of 33-amino acids from the C terminus), the isolated oxygenase domain containing the CaM binding site (OxCaM-(1-756)), and the isolated reductase domain containing the CaM binding site (RedCaM-(719 -1429)) were cloned into the NdeI and XbaI sites of the vector, pCWori ϩ (gift from Dr. M. Waterman, Vanderbilt University School of Medicine) for E. coli expression as previously described (35,36). PCR-based sitedirected mutagenesis for creation of the F584L and W587L mutants was performed with the ODA-LA PCR kit from Takara Shuzo (Osaka, Japan).…”

“…Expression and Preparation of Full-length Wild-type nNOS and the nNOS Mutants-Full-length nNOS wild-type enzyme and the mutants were expressed in the E. coli cell line BL21 (DE3), which contains plasmid pGroESL for expression of chaperone proteins, as previously described (16,33,34). Full-length nNOS wild-type and mutants (F584L, W587L, and RedCaM) were purified with DEAEToyopearl 650 M (Tosoh Co., Tokyo, Japan), 2Ј,5Ј-ADP-Sepharose and calmodulin-Sepharose column chromatography, as previously described (16,33,34).…”

“…Peptides derived from this putative autoinhibitory domain in eNOS inhibited CaM binding and Ca 2ϩ activation of both nNOS and eNOS (11). Deletion of the autoinhibitory domains of eNOS and nNOS activated the reductase domains in the absence of CaM and affected Ca 2ϩ /CaM-dependent NO formation (12)(13)(14)(15)(16). A mutant nNOS in which this region was deleted (⌬40) was 10% as active as the wild-type enzyme in the absence of Ca 2ϩ /CaM (16).…”

“…Deletion of the autoinhibitory domains of eNOS and nNOS activated the reductase domains in the absence of CaM and affected Ca 2ϩ /CaM-dependent NO formation (12)(13)(14)(15)(16). A mutant nNOS in which this region was deleted (⌬40) was 10% as active as the wild-type enzyme in the absence of Ca 2ϩ /CaM (16). C-terminal sequences found in all NOS isoforms also regulate the Ca 2ϩ /CaM-dependent electron transfers between the two flavins.…”

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

“…binding, which in turn is controlled by intracellular calcium levels [4,5,7] and is mediated by an autoinhibitory sequence in the FMN domain [19]. The oxygenase domain of the NOS isoforms receives electrons from the reductase domain in a mechanism similar to that catalysed by CPR and the cytochrome P450 enzymes.…”