Nishida C R, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
J Biol Chem. 1998 Mar 6;273(10):5566-71. doi: 10.1074/jbc.273.10.5566.
The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of .NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of .NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and L-arginine on electron transfer rates are mediated exclusively by heme domain interactions.
一氧化氮合酶（NOS）是一种单链多肽，编码一个血红素结构域、一个钙调蛋白结合基序和一个与P450还原酶具有序列相似性的黄素蛋白结构域。尽管存在这种基本的结构相似性，但三种主要的NOS同工型在其NO合成速率、细胞色素c还原速率和NADPH利用率以及这些速率对Ca2+的依赖性方面存在显著差异。为了将这些差异的起源归因于特定的蛋白质结构域，我们构建了嵌合体，其中内皮型和诱导型NOS的还原酶结构域分别被神经元型NOS的还原酶结构域所取代。嵌合蛋白的结果证实了NOS多肽链的模块化组织，并表明：（a）相似的残基在三种NOS同工型的还原酶和血红素结构域之间建立了必要的联系；（b）NO合成的最大速率由还原酶结构域向血红素结构域传递电子的最大内在能力决定；（c）诱导型NOS对Ca2+的不依赖性需要钙调蛋白与钙调蛋白结合基序和黄素蛋白结构域两者相互作用；（d）四氢生物蝶呤和L-精氨酸对电子传递速率的影响仅由血红素结构域相互作用介导。
