Regulation of the properties of the heme-NO complexes in nitric-oxide synthase by hydrogen bonding to the proximal cysteine.
Regulation of the properties of the heme-NO complexes in nitric-oxide synthase by hydrogen bonding to the proximal cysteine.
复制标题
通过与近端半胱氨酸形成氢键来调节一氧化氮合酶中血红素-NO 复合物的特性。
DOI：
10.1074/jbc.m105341200
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Rousseau,DL
中科院分区：
文献类型：
--
作者：
Couture,M;Adak,S;Stuehr,DJ;Rousseau,DL
一氧化氮合酶（NOS）催化L-精氨酸和氧生成NO和瓜氨酸。然而，已发现如此形成的NO显著地自抑制酶活性。我们假设NO反应性部分地由保守的色氨酸残基（NOS的神经元同种型（nNOS）中的位置409）和形成与血红素的近端键的半胱氨酸残基之间的氢键控制。通过使用共振拉曼光谱和NO作为血红素环境的探针，我们表明，在W 409 F和W 409 Y的神经元酶（nNOSox）的加氧酶结构域的突变体中，在Fe 3 +NO复合物中的Fe-NO键比野生型酶弱，与近端半胱氨酸残基的硫原子上的氢键的损失一致。与野生型酶相比，W 409 F和W 409 Y突变体中较弱的Fe-NO键可能导致Trp-409突变体中NO从铁血红素解离的速率更快，这可能有助于在用Trp-409突变体催化期间观察到的抑制性NO结合复合物的较低积累（Adak，S.，克鲁克斯角，王建奎，起重机，B。R.，泰纳，J.A.，Getzoff，E. D、Stuehr，D. J.（1999）J.Biol.Chem.274，26907-26911）。的光学和共振拉曼光谱的Fe 2 +NO络合物的色氨酸-409突变体不同于野生型酶的那些，并表明，一个显着的人口的五坐标Fe 2 +NO络合物是存在的。这些数据表明，由色氨酸-409残基提供的氢键是必要的，以保持硫醇盐的协调时，NO结合亚铁血红素。两者合计，我们的研究结果表明，血红素的nNOS的近端侧的环境是至关重要的形成一个稳定的铁-半胱氨酸键和血红素-NO复合物的电子性质的控制。
Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline froml-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal isoform of NOS (nNOS)) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the Trp-409 mutants as compared with the wild type enzyme, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the Trp-409 mutants (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999)J. Biol. Chem.274, 26907–26911). The optical and resonance Raman spectra of the Fe2+NO complexes of the Trp-409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate Fe2+NO complex is present. These data show that the hydrogen bond provided by the Trp-409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heme. Taken together our results indicate that the heme environment on the proximal side of nNOS is critical for the formation of a stable iron-cysteine bond and for the control of the electronic properties of heme-NO complexes.