Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer.
In neuronal nitric-oxide synthase (NOS), electron transfer proceeds across domains in a linear sequence from NADPH to flavins to heme, with calmodulin (CaM) triggering the interdomain electron transfer to the heme (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10772). Here, we utilized a neuronal NOS devoid of its bound heme and tetrahydrobiopterin (apo-NOS) to examine whether interdomain electron transfer is responsible for CaM's activation of NO synthesis, substrate-independent NADPH oxidation, and cytochrome c and ferricyanide reduction. Of the four activities, two (cytochrome c and ferricyanide reduction) were similarly stimulated by CaM in apo-NOS when compared with native NOS, indicating that activation occurs by a mechanism not involving flavin-to-heme electron transfer. Further analysis showed that CaM increased the rate of electron transfer from NADPH into the flavin centers by a factor of 20, revealing a direct activation of the NOS reductase domain by CaM. In contrast, CaM's activation of NO synthesis and substrate-independent NADPH oxidation appeared to involve flavin-to-heme electron transfer because these reactions were not activated in apo-NOS and were blocked in native NOS by agents that prevent heme iron reduction. Thus, CaM activates neuronal NOS at two points in the electron transfer sequence: electron transfer into the flavins and interdomain electron transfer between the flavins and heme. Activation at each point is associated with an up-regulation of domain-specific catalytic functions. The dual regulation by CaM is unique and represents a new means by which electron transfer can be controlled in a metalloflavoprotein.
Abu-Soud
H M
HM
Department of Immunology, Cleveland Clinic, Ohio 44195.
Yoho
L L
LL
Stuehr
D J
DJ
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000302:isolation & purification; D002147:Calmodulin / Q000502:physiology*; D002384:Catalysis; D002478:Cells, Cultured; D004579:Electron Transport; D005415:Flavins / Q000378:metabolism; D006418:Heme / Q000302:isolation & purification; D006801:Humans; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D051381:Rats
D002147:Calmodulin; D005415:Flavins; D001708:Biopterins; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin