CALMODULIN CONTROLS NEURONAL NITRIC-OXIDE SYNTHASE BY A DUAL MECHANISM - ACTIVATION OF INTRADOMAIN AND INTERDOMAIN ELECTRON-TRANSFER

In neuronal nitric-oxide synthase (NOS), electron transfer proceeds ac ross domains in a linear sequence from NADPH to flavins to heme, with calmodulin (CaM) triggering the interdomain electron transfer to the h eme (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 it s bound heme and tetrahydrobiopterin (apo NOS) to examine whether inte rdomain electron transfer is responsible for CaM's activation of NO sy nthesis, substrate-independent NADPH oxidation, and cytochrome c and f erricyanide reduction, Of the four activities, two (cytochrome c and f erricyanide reduction) were similarly stimulated by CaM in ape-NOS whe n compared with native NOS, indicating that activation occurs by a mec hanism not involving flavin-to-heme electron transfer, Further analysi s showed that CaM increased the rate of electron transfer from NADPH i nto the flavin centers by a factor of 20, revealing a direct activatio n of the NOS reductase domain by CaM. In contrast, CaM's activation of NO synthesis and substrate-independent NADPH oxidation appeared to in volve flavin-to-heme electron transfer because these reactions were no t activated in ape-NOS and were blocked in native NOS by agents that p revent heme iron reduction, Thus, CaM activates neuronal NOS at two po ints in the electron transfer sequence: electron transfer into the fla vins and interdomain electron transfer between the flavins and heme. A ctivation 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 i n a metalloflavoprotein.