Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase.
Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase.
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四氢生物蝶呤的结构调节其电子转移到一氧化氮合酶中的血红素二氧基中间体。
DOI：
10.1021/bi026898h
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Stuehr,DennisJ
中科院分区：
文献类型：
--
作者：
Wei,Chin-Chuan;Wang,Zhi-Qiang;Arvai,AndrewS;Hemann,Craig;Hille,Russ;Getzoff,ElizabethD;Stuehr,DennisJ
6R-四氢生物蝶呤（H4B）如何参与一氧化氮合酶（NOS）催化的精氨酸羟基化反应是目前研究的热点。先前对诱导型NOS的加氧酶结构域（iNOSoxy）的研究表明，H4B自由基形成在动力学上与初始血红素二氧基中间体的消失和在10 ° C下进行的单一周转反应中的Arg羟基化偶联[Wei，C. - C.的方法，王志-问：王建奎，米德，A. L.，Hemann，C.，希勒河，Stuehr，D. J.（2001）J. Biol. Chem. 276，315 - 319]。在这里，我们使用5-甲基-H4B来研究蝶呤结构如何影响自由基的形成和相关的催化步骤。在Arg存在下，5-甲基-H4B结合的iNOSoxy中的血红素二氧基中间体以35 s-1的速率反应，比H4B快3倍。这与更快的5-甲基-H4B自由基形成速率（40 vs 12.5 s-1）以及更快和更有生产力的Arg羟基化反应相结合。酶结合的5-甲基-H4B自由基的EPR谱具有不同的超精细结构比结合H4B自由基，并表现出3倍长的半衰期后，其形成。5-甲基-H4B结合的iNOSoxy的晶体结构显示，与H4B相比，结合的蝶呤的构象或其与蛋白质的相互作用的变化最小。总之，我们得出以下结论：（1）血红素二氧还原的速率与蝶呤自由基的形成有关，并且对蝶呤结构敏感。(2)更快的血红素二氧基还原增加了精氨酸羟基化的效率，但仍然限制了反应的速率。(3)5-甲基通过改变蝶呤的电子性质而不是改变蛋白质的结构或相互作用来影响血红素二氧基的还原。(4)从5-甲基-H4B的更快的电子转移可能是由于N-5甲基提供的增加的自由基稳定性。
How 6R-tetrahydrobiopterin (H4B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H4B radical formation is kinetically coupled to disappearance of an initial heme−dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 °C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001)J. Biol. Chem. 276, 315−319]. Here we used 5-methyl-H4B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme−dioxy intermediate in 5-methyl-H4B-bound iNOSoxy reacted at a rate of 35 s-1, which is 3-fold faster than with H4B. This was coupled to a faster rate of 5-methyl-H4B radical formation (40 vs 12.5 s-1) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H4B radical had different hyperfine structure than the bound H4B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H4B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H4B. Together, we conclude the following: (1) The rate of heme−dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme−dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme−dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H4B may be due to increased radical stability afforded by the N-5 methyl group.