McQuarters Ashley B, Speelman Amy L, Chen Li, Elmore Bradley O, Fan Weihong, Feng Changjian, Lehnert Nicolai
Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Biophysics, University of Michigan, Ann Arbor, MI, 48109, USA.
J Biol Inorg Chem. 2016 Dec;21(8):997-1008. doi: 10.1007/s00775-016-1396-1. Epub 2016 Sep 29.
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 HB 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, 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.
蛋白质中的第二配位层（SCS）效应受活性位点残基调控，包括氢键、静电/偶极相互作用、空间相互作用以及芳香族残基的π-堆积。在细胞色素P450中，延伸的氢键网络位于血红素近端半胱氨酸配体周围，称为“半胱氨酸口袋”。一般认为这些氢键网络调节铁-硫相互作用。先前的工作通过低温（LT）MCD光谱法鉴定了细胞色素P450cam及其相应半胱氨酸口袋突变体的高自旋（hs）三价铁形式中的S（半胱氨酸）→铁σ电荷转移跃迁[《生物化学》50:1053，2011]。在这项工作中，我们使用MCD光谱法研究了大鼠神经元型一氧化氮合酶加氧酶结构域（nNOSoxy）的高自旋三价铁状态（结合了HB和L-精氨酸）下，W409与血红素轴向半胱氨酸配体之间的氢键的影响。为此，研究了野生型酶和W409突变体，其中与轴向半胱氨酸配体的氢键网络受到了扰动。总体而言，结果与细胞色素P450cam相似，在低温MCD光谱中约27,800 cm处显示出强烈的S（半胱氨酸）→铁σ电荷转移带，表示该特征是{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对高自旋三价铁蛋白中的{血红素-硫醇盐}位点进行分类的新方法。最后，W409突变体表明，该基团的氢键对铁-硫（半胱氨酸）键强度的影响很小，至少在此处研究的蛋白质高自旋三价铁形式中如此。低温MCD光谱法用于研究W409与神经元型一氧化氮合酶血红素轴向半胱氨酸配体之间的氢键的影响。监测强烈的S（半胱氨酸）→铁σ-电荷转移带，以鉴定野生型蛋白和W409突变体中铁-硫（半胱氨酸）键的变化。
