Adak S, Ghosh S, Abu-Soud H M, Stuehr D J
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 1999 Aug 6;274(32):22313-20. doi: 10.1074/jbc.274.32.22313.
The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp(918) and Glu(919)) and one conserved aromatic residue (Phe(892)), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steady-state and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.
神经元型一氧化氮合酶（nNOS）还原酶结构域与细胞色素P450还原酶同源，后者在其黄素单核苷酸（FMN）模块中含有两个保守的酸性残基簇，这些残基在其电子转移反应中发挥着不同的作用。为了研究nNOS还原酶结构域簇1酸性残基的作用，我们对两个保守的酸性残基（天冬氨酸（Asp(918)）和谷氨酸（Glu(919)））以及一个保守的芳香族残基（苯丙氨酸（Phe(892)））进行了突变，并研究了每种突变对黄素结合、构象变化、电子转移反应、钙调蛋白调节和催化活性的影响。每种突变都会使FMN结合不稳定，但对其他方面（包括底物、辅因子或钙调蛋白结合）没有显著影响，也不会影响FMN重新组装后的催化活性，这表明突变效应仅限于FMN模块。对耗尽FMN的突变体的表征表明，结合的FMN对于nNOS血红素或细胞色素c的还原是必不可少的，但对于铁氰化物或二氯酚靛酚则不是必需的，并且确定了nNOS中的电子转移途径是从烟酰胺腺嘌呤二核苷酸磷酸（NADPH）到黄素腺嘌呤二核苷酸（FAD）再到FMN再到血红素。稳态和停流动力学分析揭示了结合的FMN在抑制NADPH对FAD的还原方面的新作用。通过去除FMN或结合钙调蛋白都可以解除这种抑制作用。钙调蛋白结合诱导了一种仅限于FMN模块的构象变化。这增加了FMN还原的速率并触发了向血红素的电子转移。我们提出，nNOS的FMN模块是nNOS中所有点处电子转移的关键正调控或负调控因子。这将nNOS与其他相关黄素蛋白区分开来，并有助于解释钙调蛋白调节的机制。
