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

在神经元型一氧化氮合酶(NNOS)中，钙调素(CaM)的结合被认为触发了从还原酶域到血红素域的电子转移，这是O2激活和NO形成所必需的。为了阐明电子传递机制，我们表征了一系列由一个全长nNOS亚基和一个加氧酶结构域亚基组成的异源二聚体。结果支持野生型nNOS的亚基间电子转移机制，即催化电子在Ca^&lt；2+&gt；/CaM依赖的方式从一个亚基的还原酶域转移到另一个亚基的血红素，正如iNOS所提出的那样。这表明这两种不同的异构体形成了相似的二聚体络合物。在一系列含有Ca^&lt；2+/CaM不敏感突变体(Delta40)的异源二聚体中，电子以不依赖于Ca^&lt；2+/CaM的方式从还原酶域转移到两种血红素。因此，在delta40突变中，从还原酶域到血红素域的电子转移可以通过亚基间和亚基内两种机制发生。然而，NO的形成活性仅与亚基间的电子转移有关，并且只有在Ca^&lt；2+&gt；/CaM存在时才能观察到。这表明CaM激活nNOS的机制并不单纯依赖于激活对nNOS血红素的电子传递，而可能涉及与血红素结构域的催化作用相关的额外结构因素。神经元型一氧化氮合酶μ是神经元型一氧化氮合酶的一种亚型，在骨骼肌细胞中特异表达，在野生型神经元型一氧化氮合酶的自身抑制区含有额外的33个氨基酸。我们成功地获得了足够数量的纯化的nNOSμ蛋白来进行鉴定。现在我们正在分析CaM和Caveoline对神经元型一氧化氮合酶μ生成NO活性的调节。

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.