Wei Chin-Chuan, Wang Zhi-Qiang, Hemann Craig, Hille Russ, Stuehr Dennis J
Department of Immunology, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
J Biol Chem. 2003 Nov 21;278(47):46668-73. doi: 10.1074/jbc.M307682200. Epub 2003 Sep 22.
Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from l-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H4B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-l-arginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy). Significant biopterin radical (>0.5 per heme) formed during reactions catalyzed by iNOSoxy that contained either H4B or 5-methyl-H4B. Biopterin radical formation was kinetically linked to conversion of a heme-dioxy intermediate to a heme-NO product complex. The biopterin radical then decayed within a 200-300-ms time period just prior to dissociation of NO from a ferric heme-NO product complex. Measures of final biopterin redox status showed that biopterin radical decay occurred via an enzymatic one-electron reduction process that regenerated H4B (or 5MeH4B). These results provide evidence of a dual redox function for biopterin during the NOHA oxidation reaction. The data suggest that H4B first provides an electron to a heme-dioxy intermediate, and then the H4B radical receives an electron from a downstream reaction intermediate to regenerate H4B. The first one-electron transition enables formation of the heme-based oxidant that reacts with NOHA, while the second one-electron transition is linked to formation of a ferric heme-NO product complex that can release NO from the enzyme. These redox roles are novel and expand our understanding of biopterin function in biology.
一氧化氮合酶是黄素血红素酶，催化两个连续的单加氧酶反应，从L-精氨酸生成一氧化氮（NO）。我们研究了酶结合辅因子6R-四氢生物蝶呤（H4B）在NO合成的第二个反应中可能的氧化还原作用，该反应是将N-羟基-L-精氨酸（NOHA）转化为NO和瓜氨酸。我们使用停流光谱和快速冷冻EPR光谱来跟踪诱导型一氧化氮合酶（iNOSoxy）加氧酶结构域催化的单周转NOHA氧化反应过程中血红素和生物蝶呤的转化。在由含有H4B或5-甲基-H4B的iNOSoxy催化的反应中形成了显著的生物蝶呤自由基（每个血红素>0.5个）。生物蝶呤自由基的形成在动力学上与血红素-双氧中间体向血红素-NO产物复合物的转化相关。然后，生物蝶呤自由基在NO从铁血红素-NO产物复合物解离之前的200-300毫秒时间内衰减。最终生物蝶呤氧化还原状态的测量表明，生物蝶呤自由基的衰减是通过酶促单电子还原过程发生的，该过程使H4B（或5MeH4B）再生。这些结果提供了生物蝶呤在NOHA氧化反应中具有双重氧化还原功能的证据。数据表明，H4B首先向血红素-双氧中间体提供一个电子，然后H4B自由基从下游反应中间体接收一个电子以再生H4B。第一次单电子转移使得能够形成与NOHA反应的基于血红素的氧化剂，而第二次单电子转移与能够从酶中释放NO的铁血红素-NO产物复合物的形成相关。这些氧化还原作用是新颖的，扩展了我们对生物蝶呤在生物学中功能的理解。
