Abstract
The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaMCC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 23111-23118 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 281 |
| Issue number | 32 |
| DOIs | |
| State | Published - Aug 11 2006 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Electron transfer by neuronal nitric-oxide synthase is regulated by concerted interaction of calmodulin and two intrinsic regulatory elements. / Roman, Linda J.; Masters, Bettie Sue S.
In: Journal of Biological Chemistry, Vol. 281, No. 32, 11.08.2006, p. 23111-23118.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Electron transfer by neuronal nitric-oxide synthase is regulated by concerted interaction of calmodulin and two intrinsic regulatory elements
AU - Roman, Linda J.
AU - Masters, Bettie Sue S
PY - 2006/8/11
Y1 - 2006/8/11
N2 - The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaMCC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.
AB - The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaMCC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.
UR - http://www.scopus.com/inward/record.url?scp=33747335659&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33747335659&partnerID=8YFLogxK
U2 - 10.1074/jbc.M603671200
DO - 10.1074/jbc.M603671200
M3 - Article
C2 - 16782703
AN - SCOPUS:33747335659
VL - 281
SP - 23111
EP - 23118
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 32
ER -