Calmodulin activates electron transfer through neuronal nitric-oxide synthase reductase domain by releasing an NADPH-dependent conformational lock
Calmodulin activates electron transfer through neuronal nitric-oxide synthase reductase domain by releasing an NADPH-dependent conformational lock
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DOI：
10.1074/jbc.m203118200
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发表时间：
2002-09-13
影响因子：
4.8
通讯作者：
Daff, S
中科院分区：
文献类型：
--
作者：
Craig, DH;Chapman, SK;Daff, S
神经元一氧化氮合酶（nNOS）是由钙调蛋白（CaM）的Ca2+依赖性结合激活的，钙调蛋白（CaM）是连接加氧酶和还原酶域的多肽连接体。钙调素结合也激活酶的还原酶结构域，增加外部电子受体（如细胞色素c）的还原速率。一些不寻常的结构特征似乎控制着这种激活机制，包括自抑制环、c端延伸和激酶依赖性磷酸化位点。nNOS还原酶结构域的预稳态还原和氧化时间过程表明，CaM结合触发NADP（+）释放，这可能对稳态转换起控制作用。此外，在没有CaM的情况下，细胞色素c还原的二级速率常数被发现高度依赖于NADPH的存在。NADPH诱导了nNOS还原酶结构域的构象变化，限制了外部电子受体进入FMN。CaM结合逆转了这种效应，导致二级速率常数增加30倍。结果表明，这两种配体之间存在着惊人的相互作用，它们都对结构域的构象施加控制，从而影响其电子转移性质。在全长酶中，NADPH结合可能会关闭体内的构象锁，阻止电子转移到加氧酶结构域，从而刺激一氧化氮的合成。
Neuronal nitric-oxide synthase (nNOS) is activated by the Ca2+-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. Presteady 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.