Exploring second coordination sphere effects in nitric oxide synthase.
Exploring second coordination sphere effects in nitric oxide synthase.
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DOI：
10.1007/s00775-016-1396-1
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发表时间：
2016-12
期刊：
影响因子：
--
通讯作者：
Lehnert N
中科院分区：
文献类型：
--
作者：
McQuarters AB;Speelman AL;Chen L;Elmore BO;Fan W;Feng C;Lehnert N
蛋白质中的第二配位球（SCS）效应由活性位点残基调节，并且包括氢键、静电/偶极相互作用、空间相互作用和芳香残基的π-堆叠。在Cyt P450中，延伸的氢键网络位于血红素的近端半胱氨酸配体周围，称为“Cys口袋”。这些氢键网络通常被认为调节Fe-S相互作用。先前的工作通过低温（LT）MCD光谱鉴定了高自旋（hs）铁形式的Cyt P450 cam和相应的Cys口袋突变体中的S（Cys）→ Fe σ CT转变[Biochemistry 50：1053，2011]。在这项工作中，我们已经调查了从W 409的轴向Cys配体的血红素在HS铁状态（与H4 B和L-Arg结合）的大鼠神经元型一氧化氮合酶加氧酶结构（nNOSoxy）使用MCD光谱的氢键的效果。为此目的，野生型酶和W 409突变体进行了研究，其中的H-键合网络与轴向Cys配体被扰动。总体而言，结果与Cyt P450 cam相似，并在LT MCD光谱中约27，800 cm−1处显示出强烈的S（Cys）→ Fe σ CT带，表明该特征是{血红素硫醇盐}活性位点的标志。这一MCD特征的发现可能构成一种新的方法来分类高铁蛋白中的{血红素-硫醇盐}位点。最后，W 409突变体表明，来自该组的氢键仅对Fe-S（Cys）键强度有很小的影响，至少在这里研究的蛋白质的hs铁形式中是如此。低温MCD光谱用于研究从W 409到血红素的轴向Cys配体的氢键在神经元型一氧化氮合酶中的作用。监测强烈的S（Cys）→ Fe σ-CT带以鉴定野生型蛋白和W 409突变体中Fe-S（Cys）键的变化
Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the ‘Cys pocket’. These hydrogen bonding networks are generally believed to regulate the Fe–S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm−1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe–S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) → Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants
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影响因子：
15
作者：
Martin, Nathaniel I.;Woodward, Joshua J.;Marletta, Michael A.
通讯作者：
Marletta, Michael A.
影响因子：
15
作者：
Cheesman, MR;Walker, FA
通讯作者：
Walker, FA
影响因子：
2.9
作者：
Ghosh, DK;Wu, CQ;Stuehr, DJ
通讯作者：
Stuehr, DJ
影响因子：
3.9
作者：
Adak, S;Stuehr, DJ
通讯作者：
Stuehr, DJ
DOI：
10.1073/pnas.87.2.682
发表时间：
1990-01-01
影响因子：
11.1
作者：
BREDT, DS;SNYDER, SH
通讯作者：
SNYDER, SH