Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer.

作者：Husam M. Abu‐Soud, Laura L. Yoho, Dennis J. Stuehr · 发表于：Journal of Biological Chemistry · 年份：1994 · DOI：10.1016/s0021-9258(18)31597-7 · 被引用次数：250 · 研究领域：Nitric Oxide and Endothelin Effects、Electrochemical sensors and biosensors、Electrochemical Analysis and Applications

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 ass...