Control of electron transfer in neuronal NO synthase.
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 Ca(2+), 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 Ca(2+) 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 flavocytochrome 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.
Daff
S
S
Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K. Simon.Daff@ed.ac.uk
Noble
M A
MA
Craig
D H
DH
Rivers
S L
SL
Chapman
S K
SK
Munro
A W
AW
Fujiwara
S
S
Rozhkova
E
E
Sagami
I
I
Shimizu
T
T
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D002147:Calmodulin / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002147:Calmodulin; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D009249:NADP; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases