Study for the regulation mechanism of nitric oxide synthase activity by an autoinhibitory domain and calmodulin

在神经元一氧化氮合酶（nNOS）中，钙调素（CaM）结合被认为触发电子从还原酶结构域转移到血红素结构域，这是O2激活和NO形成所必需的。为了阐明电子转移机制，我们对一系列由一个全长nNOS亚基和一个氧化酶结构域亚基组成的异源二聚体进行了表征。结果支持野生型nNOS的亚基间电子转移机制，因为催化电子以Ca^<2+>/CaM依赖的方式从一个亚基的还原酶结构域转移到另一个亚基的血红素，正如iNOS所提出的那样。这表明两种不同的异构体形成相似的二聚体复合物。在一系列含有Ca^<2+>/CaM不敏感突变体（delta40）的异源二聚体中，电子以Ca^<2+>/CaM独立的方式从还原酶结构域转移到两个血红素。因此，在delta40突变体中，从还原酶结构域到血红素结构域的电子转移可以通过亚基间和亚基内机制发生。然而，NO的形成活性仅与亚基间电子转移有关，并且仅在Ca^<2+>/CaM存在时观察到。这表明CaM活化nNOS的机制不仅仅依赖于激活电子向nNOS血红素的转移，还可能涉及与血红素结构域催化作用相关的其他结构因素。接下来，我们构建了nNOSμ在大肠杆菌细胞中的表达体系。nNOSμ是nNOS的异构体，在骨骼肌细胞中特异性表达，在野生型nNOS的自抑制结构域中含有额外的33个氨基酸。我们成功地获得了足够量的纯化nNOSμ蛋白来进行鉴定。目前我们正在分析CaM和小泡碱对nNOSμ细胞NO生成活性的调节作用。

In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O2 activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild-type nNOS, in that electrons for catalysis transfer in a Ca^<2+>/CaM dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for iNOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca^<2+>/CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca^<2+>/CaM independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca^<2+>/CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes, but may involve additional structural factors linked to the catalytic action of the heme domain.Next we constructed an expression system of nNOSμ in E. coli cell. nNOSμ is an isoform of nNOS which specifically expressed in skeletal muscle cells and contains extra-33 amino acids in the autoinhibitory domain of nNOS wild-type. We succeeded to get enough amount of purified nNOSμ protein to characterize. Now we are undergoing to analyze the regulation of NO formation activity of nNOSμ by CaM and caveoline.