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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component
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Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component

机译：组装支架NifEN：固氮酶催化成分的结构和功能同源物

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摘要

NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster–bound NifEN (designated NifENM) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifENM upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifENM with an M-cluster in one αβ-half and an empty cluster-binding site in the other αβ-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αβ-dimers of NifEN. Perhaps most importantly, NifENM displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster–bound αβ-dimer of NifENM as a structural and functional mimic of one catalytic αβ-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.
机译：NifEN是Mo固氮酶（称为M簇）的辅助因子的生物合成支架。先前的研究已经揭示了NifEN和NifDK（固氮酶的催化成分）之间的序列和结构同源性。然而，直接证明这两种蛋白质之间的功能同源性仍然难以捉摸。在这里，我们表明，在NifEN上辅因子前体（称为L-簇）成熟后，从NifEN提取的簇物种在光谱上等效，并且在功能上可与从NifDK提取的天然M-簇互换。这两个提取的团簇均显示出几乎不可区分的EPR特征，X射线吸收光谱/扩展的X射线吸收精细结构（XAS / EXAFS）光谱和重构活性，从而牢固地建立了与M簇结合的NifEN（称为NifEN M ）是除NifDK之外唯一包含独特固氮酶辅因子的蛋白质。铁螯合实验表明，成熟后簇从NifEN M 中的表面重定位到其结合位点，这与将M-簇插入NifDK中类似的结合位点相似，而金属分析表明， NifEN M 的一个不对称构象，其中一个αβ-一半具有一个M-簇，而另一个αβ-一半具有一个空的簇结合位点，这导致提出了一种M-逐步组装机制的提议。在NifEN的两个αβ-二聚体中聚集。也许最重要的是，NifEN M 与NifDK具有相似的ATP依赖性底物还原特征，从而确定了NifEN M 的M-簇结合的αβ-二聚体。 NifDK的一个催化性αβ-一半的结构和功能模拟，同时表明该蛋白作为进一步研究固氮酶机理的有用工具的潜力。

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