Gao Ying Tong, Panda Satya Prakash, Roman Linda J, Martásek Pavel, Ishimura Yuzuru, Masters Bettie Sue S
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
J Biol Chem. 2007 Mar 16;282(11):7921-9. doi: 10.1074/jbc.M609814200. Epub 2007 Jan 17.
Nitric-oxide synthases (NOS) catalyze nitric oxide (NO) formation from the amino acid L-arginine. NOS is known to catalyze more than one reaction: the NO-producing reaction is considered to be the coupled reaction, and the uncoupled reactions are those that produce reactive (reduced) oxygen species (ROS), such as superoxide anion (O-2.) and/or hydrogen peroxide (H2O2). As an oxygenase, NOS has been known for more than two decades, yet there is no complete description of oxygen stoichiometry. The present paper is focused on oxygen stoichiometry and the effects of cofactor binding on the neuronal isoform (nNOS) on oxygen uptake and product formation. Products of the uncoupled reactions are analyzed using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for both O-2. and H2O2. The addition of calmodulin not only stimulated the oxygen uptake rate but also changed the product of the uncoupled reaction, supporting the possibility of two different sites for electron leakage to molecular oxygen. Quantitative analysis of the uncoupled (substrate-free) reaction revealed a stoichiometry close to the theoretical value, and adding L-arginine not only initiates the coupled reaction, but also inhibits oxygen uptake. The presence of tetrahydrobiopterin affects oxygen metabolism by lowering the apparent Km value of nNOS for oxygen in the uncoupled reaction.
一氧化氮合酶（NOS）催化从氨基酸L-精氨酸生成一氧化氮（NO）。已知NOS催化不止一种反应：产生NO的反应被认为是偶联反应，而非偶联反应则是产生活性（还原型）氧物种（ROS）的反应，如超氧阴离子（O₂⁻）和/或过氧化氢（H₂O₂）。作为一种加氧酶，NOS已被知晓二十多年了，但氧化学计量学尚无完整描述。本文聚焦于氧化学计量学以及辅因子结合对神经元亚型（nNOS）的氧摄取和产物形成的影响。使用二乙酰氘代血红素取代的辣根过氧化物酶作为O₂⁻和H₂O₂的捕获剂来分析非偶联反应的产物。钙调蛋白的添加不仅刺激了氧摄取速率，还改变了非偶联反应的产物，这支持了电子向分子氧泄漏存在两个不同位点的可能性。对非偶联（无底物）反应的定量分析揭示了一种接近理论值的化学计量关系，添加L-精氨酸不仅启动了偶联反应，还抑制了氧摄取。四氢生物蝶呤的存在通过降低非偶联反应中nNOS对氧的表观Km值来影响氧代谢。
