Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site.
Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site.
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DOI：
10.1039/d1dt02219a
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发表时间：
2021-09-21
期刊：
影响因子：
--
通讯作者：
Vincent KA
中科院分区：
文献类型：
--
作者：
Morra S;Duan J;Winkler M;Ash PA;Happe T;Vincent KA
阐明氧化还原金属酶活性位点的中间体分布对于理解其高效催化作用至关重要。在这里，我们证明可以在蛋白质晶体中生成并检测 [FeFe]-氢化酶的关键催化氧化还原态。来自巴氏梭菌 (CpI) 的原型 [FeFe]-氢化酶 I 的单个晶体保持在电化学控制下，从而可以精确调节氧化还原电位，同时通过傅里叶变换红外 (FTIR) 显微光谱对晶体进行探测。高信号/噪声光谱揭示了根据内源 CO 和 CN− 配体的状态特异性振动带，活性位点（H 团簇）氧化还原态分布的电势依赖性变化。 CpI 晶体显示出与溶液中检测到的相同的 H 团簇状态，包括氧化物质 Hox、还原物质 Hred/HredH+、超还原 HsredH+ 和氢化物物质 Hhyd。这种方法提供的高灵敏度和精确的氧化还原控制还有助于检测和表征仅在狭窄的条件窗口内积累的低丰度物种，从而揭示新的氧化还原中间体。 [FeFe]-氢化酶单晶中氧化还原物质的分布可以通过电化学方式进行调节。所有催化中间体均通过同步红外显微光谱观察，为富集特定氧化还原态提供了路线图。
Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and detect, the key catalytic redox states of an [FeFe]-hydrogenase in a protein crystal. Individual crystals of the prototypical [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultaneously probed via Fourier Transform Infrared (FTIR) microspectroscopy. The high signal/noise spectra reveal potential-dependent variation in the distribution of redox states at the active site (H-cluster) according to state-specific vibrational bands from the endogeneous CO and CN− ligands. CpI crystals are shown to populate the same H-cluster states as those detected in solution, including the oxidised species Hox, the reduced species Hred/HredH+, the super-reduced HsredH+ and the hydride species Hhyd. The high sensitivity and precise redox control offered by this approach also facilitates the detection and characterisation of low abundance species that only accumulate within a narrow window of conditions, revealing new redox intermediates. The distribution of redox species in single crystals of [FeFe]-hydrogenase can be tuned electrochemically. All catalytic intermediates are observed by simultaneous infrared microspectroscopy, providing a roadmap for enriching specific redox states.
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影响因子：
8.4
作者：
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影响因子：
18.3
作者：
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通讯作者：
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