Ash Philip A, Hidalgo Ricardo, Vincent Kylie A
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory.
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory;
J Vis Exp. 2017 Dec 4(130):55858. doi: 10.3791/55858.
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.
了解氧化还原蛋白的化学性质需要能够精确控制蛋白内氧化还原中心的方法。蛋白质膜电化学技术是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，该技术为一系列不同蛋白质的氧化还原反应提供了功能方面的见解。要全面理解其化学性质，需要将电化学控制与其他能够提供更多结构和机理见解的技术相结合。在此，我们展示一种技术——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比表面积炭黑电极上的氧化还原蛋白。将此电极集成到流通池中，可在测量过程中改变溶液的pH值或溶质浓度。这在研究氧化还原酶时特别有用，因为在电极上可以维持并控制快速的催化周转，从而能够对催化机制中长寿命中间体进行光谱观察。我们通过在大肠杆菌氢化酶1的周转（H2氧化）和非周转条件下进行实验来展示该技术。
