A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin.
A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin.
复制标题
一氧化氮合酶中的保守色氨酸通过四氢生物蝶呤调节血红素二氧还原。
DOI：
10.1021/bi011182s
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Stuehr,DJ
中科院分区：
文献类型：
--
作者：
Wang,ZQ;Wei,CC;Ghosh,S;Meade,AL;Hemann,C;Hille,R;Stuehr,DJ
在一氧化氮合酶（NOS）中，（6 R）-四氢生物蝶呤（H4 B）在血红素附近结合，并且可以在Arg羟基化期间还原血红素-二氧中间体（FeIIO 2）[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]。一个保守的Trp与H4 B进行芳香堆积，其突变抑制NO合成。为了研究这种W 457如何影响H4 B氧化还原功能，我们用小鼠诱导型NOS加氧酶结构域（iNOSoxy）突变体W 457 F和W 457 A进行了单周转反应。将含Arg和H4 B的亚铁突变体与含O2的缓冲液混合，然后跟踪血红素光谱跃迁、H4 B自由基形成和Arg羟基化随时间的变化。在W 457 A和W 457 F中观察到血红素FeIIO 2中间体，并具有正常的光谱特征。但其消失速度（W 457 F为6.5 s-1，W 457 A为3.0 s-1）比野生型（12.5 s-1）慢。H4 B自由基形成速率（W 457 F为7.1 s-1，W 457 A为2.7 s-1）与FeIIO 2消失速率相匹配，但比野生型自由基形成速率（13 s-1）慢。突变体中H4 B自由基形成的程度与野生型相似，但它们的自由基衰变快2 - 4倍。这些动力学变化与突变体（野生型> W 457 F> W 457 A）的较慢和较不广泛的Arg羟基化相关。我们的结论是，W 457确保了正确的克里思的电子转移从H4 B血红素FeIIO 2，可能通过稳定H4 B自由基。这些参数的适当控制可能有助于最大限度地提高精氨酸羟基化和最大限度地减少非偶联O2在血红素激活。
In nitric oxide synthase (NOS), (6R)-tetrahydrobiopterin (H4B) binds near the heme and can reduce a heme−dioxygen intermediate (FeIIO2) during Arg hydroxylation [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]. A conserved Trp engages in aromatic stacking with H4B, and its mutation inhibits NO synthesis. To examine how this W457 impacts H4B redox function, we performed single turnover reactions with the mouse inducible NOS oxygenase domain (iNOSoxy) mutants W457F and W457A. Ferrous mutants containing Arg and H4B were mixed with O2-containing buffer, and then heme spectral transitions, H4B radical formation, and Arg hydroxylation were followed versus time. A heme FeIIO2intermediate was observed in W457A and W457F and had normal spectral characteristics. However, its disappearance rate (6.5 s-1in W457F and 3.0 s-1in W457A) was slower than in wild-type (12.5 s-1). Rates of H4B radical formation (7.1 s-1in W457F and 2.7 s-1in W457A) matched their rates of FeIIO2disappearance, but were slower than radical formation in wild-type (13 s-1). The extent of H4B radical formation in the mutants was similar to wild-type, but their radical decayed 2−4 times faster. These kinetic changes correlated with slower and less extensive Arg hydroxylation by the mutants (wild-type > W457F > W457A). We conclude that W457 ensures a correct tempo of electron transfer from H4B to heme FeIIO2, possibly by stabilizing the H4B radical. Proper control of these parameters may help maximize Arg hydroxylation and minimize uncoupled O2activation at the heme.