NEET蛋白构成了一类独特的[2Fe-2S]蛋白。金属离子与三个半胱氨酸和一个组氨酸结合。蛋白质簇存在于两种氧化还原状态;氧化的蛋白质(含有两个FeIII离子)可以将簇转移到apo-受体蛋白质,而还原形式(包含一个亚铁离子)仍然与蛋白质框架结合。这里,我们对还原和氧化形式的人类NEET蛋白进行了计算机模拟和体外研究。对所有可用的人类NEET蛋白质结构的量子化学计算表明,减少簇削弱了Fe-NHis和Fe-SCys键,与其他Fe-S蛋白中看到的类似(例如,铁氧还蛋白和Rieske蛋白)。我们进一步表明,NEET蛋白之一(mNT)的[2Fe-2S]簇中的额外电子位于His结合的铁离子上,与我们以前的光谱研究一致。动力学测量表明mNT[2Fe-2S]+仅通过温度升高而释放。因此,人NEET蛋白[2Fe-2S]簇的还原状态在动力学上是惰性的。这种以前未认识到的还原状态的动力学惰性,随着氧化态的反应性,在所有[2Fe-2S]蛋白质中都是独一无二的。最后,使用共同进化分析,以及分子动力学模拟,我们提供了关于在环L2和簇区域之间观察到的变形物的见解。具体来说,我们表明,W75,R76,K78,K79,F82和G85在后一个区域在两种氧化还原状态下具有相似的变构特征。
The NEET proteins constitute a unique class of [2Fe-2S] proteins. The metal ions bind to three cysteines and one histidine. The proteins\' clusters exist in two redox states; the oxidized protein (containing two FeIII ions) can transfer the cluster to apo-acceptor protein(s), while the reduced form (containing one ferrous ion) remains bound to the protein frame. Here, we perform in silico and in vitro studies on human NEET proteins in both reduced and oxidized forms. Quantum chemical calculations on all available human NEET proteins structures suggest that reducing the cluster weakens the Fe-NHis and Fe-SCys bonds, similar to what is seen in other Fe-S proteins (e.g., ferredoxin and Rieske protein). We further show that the extra electron in the [2Fe-2S]+ clusters of one of the NEET proteins (mNT) is localized on the His-bound iron ion, consistently with our previous spectroscopic studies. Kinetic measurements demonstrate that the mNT [2Fe-2S]+ is released only by an increase in temperature. Thus, the reduced state of human NEET proteins [2Fe-2S] cluster is kinetically inert. This previously unrecognized kinetic inertness of the reduced state, along with the reactivity of the oxidized state, is unique across all [2Fe-2S] proteins. Finally, using a coevolutionary analysis, along with molecular dynamics simulations, we provide insight on the observed allostery between the loop L2 and the cluster region. Specifically, we show that W75, R76, K78, K79, F82 and G85 in the latter region share similar allosteric characteristics in both redox states.