Du Mei, Yeh Hui-Chun, Berka Vladimir, Wang Lee-Ho, Tsai Ah-Lim
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
J Biol Chem. 2003 Feb 21;278(8):6002-11. doi: 10.1074/jbc.M209606200. Epub 2002 Dec 11.
Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e. eNOS(ox) and eNOS(red), has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS(ox) and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOS(ox) were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS(ox) domain. Stoichiometric titration of eNOS(ox) demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5. Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOS(ox). Ferrous eNOS(ox), in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements. Calmodulin binds eNOS(red) at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH(4) radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS.
内皮型一氧化氮合酶（eNOS）氧化还原特性的表征对于理解这种参与心血管功能的重要酶的复杂反应机制至关重要。为实现这一目标，已建立人eNOS氧化酶和还原酶结构域（即eNOS(ox)和eNOS(red)）在酵母中的过表达。对静息态eNOS(ox)及其与各种配体的复合物进行紫外可见光谱和电子顺磁共振（EPR）光谱表征，表明其为典型的NOS血红素结构，即硫醇盐血红素蛋白。EPR解析出两个低自旋咪唑血红素复合物，但未解析出分离的eNOS(ox)，这表明二聚体eNOS(ox)结构域的血红素几何结构存在细微差异。eNOS(ox)的化学计量滴定表明，血红素具有1 - 1.5个还原当量的容量。在血红素还原发生之前，还消耗了额外的1.5 - 2.5个还原当量，这表明存在其他未知的高电位氧化还原中心。没有迹象表明存在其他金属中心可以解释eNOS(ox)的这种额外电子容量。如快速冷冻EPR测量所示，在L - 精氨酸存在下，亚铁eNOS(ox)在与氧气混合时完全能够形成四氢生物蝶呤自由基。钙调蛋白以1:1的化学计量比和高亲和力结合eNOS(red)。化学计量滴定和计算机模拟能够确定FMN和FAD四个半反应之间的三个氧化还原电位分离。还可以在多个波长下解析每种黄素的半醌、氧化态和还原态的消光系数。通过化学计量滴定对eNOS两个结构域进行的首次氧化还原表征以及为BH(4)自由基中间体生成高质量的EPR光谱，说明了这些工具在未来对eNOS反应机制进行详细研究中的有用性。
