Characterization of [4Fe-4S] cluster vibrations and structure in nitrogenase Fe protein at three oxidation levels via combined NRVS, EXAFS, and DFT analyses.
Characterization of [4Fe-4S] cluster vibrations and structure in nitrogenase Fe protein at three oxidation levels via combined NRVS, EXAFS, and DFT analyses.
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DOI：
10.1021/ja307027n
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发表时间：
2013-02-20
影响因子：
15
通讯作者：
Cramer SP
中科院分区：
文献类型：
--
作者：
Mitra D;George SJ;Guo Y;Kamali S;Keable S;Peters JW;Pelmenschikov V;Case DA;Cramer SP
棕色固氮菌固氮酶Fe蛋白（Av 2）提供了一个难得的机会，在同一蛋白质环境中研究三个氧化水平的[4Fe-4S]簇。在这里，我们报告的结构和振动的变化后，减少使用NRVS和EXAFS光谱和DFT计算相结合的集群。这项工作的关键是这三种技术之间的协同作用，因为每种技术都产生高度互补的信息，而且它们的分析方法是相互依存的。重要的是，光谱样品不含玻璃化剂。NRVS和DFT揭示了Fe-S伸缩频率随每个电子的增加而系统地降低10-30 cm−1。“氧化”的[4Fe-4S]2+态光谱与以前的共振拉曼光谱一致并扩展了以前的共振拉曼光谱。对于Fe蛋白中的“还原”[4Fe-4S]1+态和任何“全铁”[4Fe-4S]0簇，这些NRVS光谱是第一个可用的振动数据。NRVS模拟也允许估计的Fe-S和Fe-Fe距离的振动无序，限制EXAFS分析，并允许结构无序估计。对于氧化的Av 2，EXAFS和DFT表明几乎相等的Fe-Fe距离，而添加一个电子降低了簇的对称性。然而，添加第二个电子以形成全铁态会引起显着的结构变化。EXAFS数据记录到k = 21 <$−1，表明在2.56和2.75 <$的Fe-Fe相互作用比为1：1，这一结果与DFT一致。破缺对称性（BS）DFT合理化氧化还原状态和Fe-S和Fe-Fe距离之间的相互作用，作为[4Fe-4S]簇固有的主要自旋依赖行为，并受到Av 2蛋白质环境的干扰。
Azotobacter vinelandii nitrogenase Fe protein (Av2) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same protein environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies and DFT calculations. Key to this work is the synergy between these three techniques as each generates highly complementary information and their analytical methodologies are interdependent. Importantly, the spectroscopic samples contained no glassing agents. NRVS and DFT reveal a systematic 10-30 cm−1 decrease in Fe-S stretching frequencies with each added electron. The “oxidized” [4Fe-4S]2+ state spectrum is consistent with and extends previous resonance Raman spectra. For the “reduced” [4Fe-4S]1+ state in Fe protein, and for any “all-ferrous” [4Fe-4S]0 cluster, these NRVS spectra are the first available vibrational data. NRVS simulations also allow estimation of the vibrational disorder for Fe-S and Fe-Fe distances, constraining the EXAFS analysis and allowing structural disorder to be estimated. For oxidized Av2, EXAFS and DFT indicate nearly equal Fe-Fe distances, while addition of one electron decreases the cluster symmetry. However, addition of the second electron to form the all-ferrous state induces significant structural change. EXAFS data recorded to k = 21 Å−1 indicates a 1:1 ratio of Fe-Fe interactions at 2.56 Å and 2.75 Å, a result consistent with DFT. Broken symmetry (BS) DFT rationalizes the interplay between redox state and the Fe-S and Fe-Fe distances as predominantly spin-dependent behavior inherent to the [4Fe-4S] cluster and perturbed by the Av2 protein environment.
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