Interactions between the isolated oxygenase and reductase domains of neuronal nitric-oxide synthase: assessing the role of calmodulin.
Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca(2+)/calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM. N(G)-hydroxy-l-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates for N(G)-hydroxy-l-Arg in the absence of Ca(2+)/CaM in most of the reconstituted systems were 2.3-3.1 min(-1). Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca(2+)/CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems.
Rozhkova
Elena A
EA
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
Fujimoto
Norikazu
N
Sagami
Ikuko
I
Daff
Simon N
SN
Shimizu
Toru
T
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000502:physiology*; D002384:Catalysis; D003574:Cytochrome c Group / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002147:Calmodulin; D003574:Cytochrome c Group; D018076:DNA, Complementary; D006418:Heme; D009249:NADP; D010088:Oxidoreductases; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D009251:NADPH-Ferrihemoprotein Reductase; D010100:Oxygen