Panda K, Ghosh S, Stuehr D J
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 2001 Jun 29;276(26):23349-56. doi: 10.1074/jbc.M100687200. Epub 2001 Apr 26.
Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH. Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups. We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer. A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as full-length subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation. The E592A mutation prevented Arg binding to the oxygenase domain in which it was present. It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit. However, it had no effect when present in the full-length subunit (i.e. the subunit containing the reductase domain). The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin. This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS. In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.
神经元型一氧化氮合酶（nNOS）由一个结合血红素、（6R）-四氢生物蝶呤和精氨酸的加氧酶结构域与一个结合黄素腺嘌呤二核苷酸（FAD）、黄素单核苷酸（FMN）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的还原酶结构域组成。其活性需要两个加氧酶结构域之间的二聚体相互作用以及还原酶和加氧酶结构域之间的钙调蛋白结合，这会触发黄素和血红素基团之间的电子转移。我们构建了四种不同的nNOS异二聚体，以确定钙调蛋白诱导的电子转移在nNOS二聚体中的路径。大鼠nNOS的一个主要为单体的突变体（G671A）及其精氨酸结合突变体（G671A/E592A）被用作全长亚基，同时使用了含有或不含有E592A突变的加氧酶结构域伙伴。E592A突变阻止了精氨酸与存在该突变的加氧酶结构域结合。当它位于与全长亚基相邻的加氧酶结构域中时，也会阻止一氧化氮（NO）的合成。然而，当它存在于全长亚基（即含有还原酶结构域的亚基）中时则没有影响。活性异二聚体（G671A/E592A全长亚基加野生型加氧酶结构域亚基）在对五种不同形式和钙调蛋白嵌合体的催化反应中与野生型同二聚体nNOS表现出显著相似性。这揭示了钙调蛋白在支持nNOS相邻亚基上的黄素和血红素基团之间的跨亚基电子转移中发挥着积极作用。总之，我们提出钙调蛋白的作用是使nNOS二聚体中相邻的还原酶和加氧酶结构域正确对齐，以便它们之间进行电子转移，从而导致血红素合成NO。
