The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca2+, whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40–50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca2+ concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. The results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement. We also report the creation of chimaeric enzymes in which the reductase domains of nNOS and flavo-cytochrome P450 BM3 (Bacillus megaterium III) have been exchanged. Despite its very different flavin redox potentials, the BM3 reductase domain was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.
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Conference Article|
May 01 2001
Control of electron transfer in neuronal NO synthase
S. Daff;
S. Daff
1
*Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K.
1To whom correspondence should be addressed (e-mail Simon.Daff@ed.ac.uk)
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M. A. Noble;
M. A. Noble
*Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K.
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D. H. Craig;
D. H. Craig
*Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K.
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S. L. Rivers;
S. L. Rivers
*Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K.
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S. K. Chapman;
S. K. Chapman
*Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K.
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A. W. Munro;
A. W. Munro
†Department of Pure & Applied Chemistry, University of Strathclyde, Glasgow, U.K.
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S. Fujiwara;
S. Fujiwara
‡Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
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E. Rozhkova;
E. Rozhkova
‡Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
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I. Sagami;
I. Sagami
‡Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
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T. Shimizu
T. Shimizu
‡Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
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Publisher: Portland Press Ltd
Received:
November 15 2000
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2001 Biochemical Society
2001
Biochem Soc Trans (2001) 29 (2): 147–152.
Article history
Received:
November 15 2000
Citation
S. Daff, M. A. Noble, D. H. Craig, S. L. Rivers, S. K. Chapman, A. W. Munro, S. Fujiwara, E. Rozhkova, I. Sagami, T. Shimizu; Control of electron transfer in neuronal NO synthase. Biochem Soc Trans 1 May 2001; 29 (2): 147–152. doi: https://doi.org/10.1042/bst0290147
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