Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii
Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii
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DOI：
10.1021/jacs.2c13458
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发表时间：
2023-05-10
影响因子：
15
通讯作者：
Silakov，Alexey
中科院分区：
文献类型：
--
作者：
Corrigan，Patrick S.;Majer，Sean H.;Silakov，Alexey
贝氏梭状芽孢杆菌氢酶(CbHydA1)是一种不寻常的氢酶，它可以通过可逆地转变为O2保护的非活性状态(Hinact)来承受长时间的O2暴露。过去已经表明，在六铁活性中心(H-簇)的[2Fe]H部分附近的“SC367CP”环的非典型构象允许Cys367残基采用“脱离-H+-途径”的取向，促进辅因子向Hinact的转变。在这里，我们研究了在氧化条件下直接转化为H原子的氧化态(HOX)和相关的CO抑制态(HOX-CO)的电子结构。我们证明了这两种状态在电子顺磁共振(EPR)谱中表现出两种不同的形式。这两种形式之间的比例与pH有关，但对缓冲液的选择也很敏感。红外光谱和电子顺磁共振分析表明，光谱的异质性是由于H-团簇的[4Fe4S]H子团簇的配位环境发生了微扰，而没有影响[2Fe]H子团簇。总体而言，我们得出的结论是，每个状态下两个光谱分量的观测是H团簇环境异质性的证据，这可能与SCCP环的构象迁移率有关。这种灵活性可能允许Cys367在关闭H+途径和开启H+途径的旋转子之间快速切换。因此，我们认为这种结构流动性可能是保持高酶活性同时允许轻松过渡到O2保护状态的关键。
[FeFe] hydrogenase fromClostridium beijerinkii(CbHydA1) is an unusual hydrogenase in that it can withstand prolonged exposure to O2by reversibly converting into an O2-protected, inactive state (Hinact). It has been indicated in the past that an atypical conformation of the “SC367CP” loop near the [2Fe]Hportion of the six-iron active site (H-cluster) allows the Cys367 residue to adopt an “off-H+-pathway” orientation, promoting a facile transition of the cofactor to Hinact. Here, we investigated the electronic structure of the H-cluster in the oxidized state (Hox) that directly converts to Hinactunder oxidizing conditions and the related CO-inhibited state (Hox-CO). We demonstrate that both states exhibit two distinct forms in electron paramagnetic resonance (EPR) spectroscopy. The ratio between the two forms is pH-dependent but also sensitive to the buffer choice. Our IR and EPR analyses illustrate that the spectral heterogeneity is due to a perturbation of the coordination environment of the H-cluster’s [4Fe4S]Hsubcluster without affecting the [2Fe]Hsubcluster. Overall, we conclude that the observation of two spectral components per state is evidence of heterogeneity of the environment of the H-cluster likely associated with conformational mobility of the SCCP loop. Such flexibility may allow Cys367 to switch rapidly between off- and on-H+-pathway rotamers. Consequently, we believe such structural mobility may be the key to maintaining high enzymatic activity while allowing a facile transition to the O2-protected state.