Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase.
Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase.
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DOI：
10.3791/55858
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发表时间：
2017-12-04
期刊：
影响因子：
--
通讯作者：
Vincent KA
中科院分区：
文献类型：
--
作者：
Ash PA;Hidalgo R;Vincent KA
了解氧化还原蛋白的化学性质需要能够精确控制蛋白质内氧化还原中心的方法。蛋白质膜电化学技术将蛋白质固定在电极表面，使电极取代生理电子供体或受体，为一系列不同蛋白质的氧化还原反应提供了功能性的见解。全面的化学理解需要将电化学控制与其他技术相结合，以增加额外的结构和机械洞察力。在这里，我们展示了一种技术，即蛋白质膜红外电化学，它将蛋白质膜电化学与氧化还原蛋白的红外光谱采样相结合。该技术使用多重反射衰减全反射几何结构来探测固定在高表面积炭黑电极上的氧化还原蛋白。将该电极合并到流通池中可以在测量过程中改变溶液的 pH 值或溶质浓度。这在处理氧化还原酶方面特别强大，可以在电极上维持和控制快速的催化周转，从而可以对催化机制中长寿命的中间物质进行光谱观察。我们通过在周转（H2 氧化）和非周转条件下对大肠杆菌氢化酶 1 进行实验来演示该技术。
Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.
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影响因子：
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