Ilagan Robielyn P, Tiso Mauro, Konas David W, Hemann Craig, Durra Deborah, Hille Russ, Stuehr Dennis J
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 2008 Jul 11;283(28):19603-15. doi: 10.1074/jbc.M802914200. Epub 2008 May 16.
Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.
一氧化氮（NO）是由一氧化氮合酶（NOS）合成的一种生理介质。尽管它们在结构上相似，但内皮型一氧化氮合酶（eNOS）的NO合成活性比神经元型一氧化氮合酶（nNOS）低6倍，细胞色素c还原酶活性低6至16倍，这意味着它们的电子转移能力存在显著差异。我们利用这两种酶的纯化还原酶结构域构建体（牛eNOSr和大鼠nNOSr）来研究以下三种可能控制其电子转移的机制：（i）其FMN亚结构域的双态构象平衡的设定点和控制；（ii）黄素中点还原电位；（iii）NOSr-NADP+相互作用的动力学。尽管eNOSr和nNOSr在NADP(H)相互作用和黄素热力学方面存在差异，但这些差异很小，不太可能解释它们不同的电子转移活性。相比之下，当结合的FMN辅因子处于其三种可能的氧化态中的每一种时，无钙调蛋白（CaM）的eNOSr比无CaM的nNOSr更倾向于FMN屏蔽（电子接受）构象而非FMN去屏蔽（电子供体）构象。NADPH结合仅稳定了nNOSr的FMN屏蔽构象，而CaM使两种酶都向FMN去屏蔽构象转变。对第一次催化周转期间测得的细胞色素c还原速率的分析表明，eNOSr和nNOSr向FMN去屏蔽状态的构象变化速率不同，并且是无CaM酶的限速因素。我们得出结论，eNOSr和nNOSr中FMN构象平衡的设定点和调节存在显著差异，这可以解释eNOSr较低的电子转移活性。
