Panda Koustubh, Haque Mohammad Mahfuzul, Garcin-Hosfield Elsa D, Durra Deborah, Getzoff Elizabeth D, Stuehr Dennis J
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 2006 Dec 1;281(48):36819-27. doi: 10.1074/jbc.M606129200. Epub 2006 Sep 25.
The FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu(762). Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis.
一氧化氮合酶（NOS）的黄素单核苷酸（FMN）模块发挥着关键作用，它将烟酰胺腺嘌呤二核苷酸磷酸（NADPH）衍生的电子转移至酶血红素，以供氧气活化之用。该过程可能涉及一种摆动机制，即FMN模块的同一面在催化过程中接受并提供电子。晶体结构显示，FMN模块的这一面呈电负性，而互补的相互作用表面呈电正性，这意味着电荷相互作用促成了其功能。我们运用定点诱变技术，研究了FMN模块六个带负电表面残基在神经元型NOS电子转移和催化过程中的作用。研究结果结合NOS及相关黄素蛋白的晶体结构进行解读。中和或反转每个残基的负电荷，会改变神经元型NOS的一氧化氮合成、NADPH氧化酶及细胞色素c还原酶活性，还会改变血红素还原。最大的影响出现在NOS特有的带电荷残基谷氨酸（Glu）762处。总体而言，这些结果表明，FMN模块的静电相互作用有助于调节电子转移，并在NOS催化过程中使黄素自氧化及活性氧生成降至最低。
