Calmodulin activates electron transfer through neuronal nitric-oxide synthase reductase domain by releasing an NADPH-dependent conformational lock.
Neuronal nitric-oxide synthase (nNOS) is activated by the Ca(2+)-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP(+) release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.
Craig
Daniel H
DH
Department of Chemistry, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Chapman
Stephen K
SK
Daff
Simon
S
D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
D002147:Calmodulin; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat