Léger Christophe, Elliott Sean J, Hoke Kevin R, Jeuken Lars J C, Jones Anne K, Armstrong Fraser A
Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QR, UK.
Biochemistry. 2003 Jul 29;42(29):8653-62. doi: 10.1021/bi034789c.
Protein film voltammetry is a relatively new approach to studying redox enzymes, the concept being that a sample of a redox protein is configured as a film on an electrode and probed by a variety of electrochemical techniques. The enzyme molecules are bound at the electrode surface in such a way that there is fast electron transfer and complete retention of the chemistry of the active site that is observed in more conventional experiments. Modulations of the electrode potential or catalytic turnover result in the movement of electrons to, from, and within the enzyme; this is detected as a current that varies in characteristic ways with time and potential. Henceforth, the potential dimension is introduced into enzyme kinetics. The presence of additional intrinsic redox centers for providing fast intramolecular electron transfer between a buried active site and the protein surface is an important factor. Centers which carry out cooperative two-electron transfer, most obviously flavins, produce a particularly sharp signal that allows them to be observed, even as transient states, when spectroscopic methods are not useful. High catalytic activity produces a large amplification of the current, and useful information can be obtained even if the coverage on the electrode is low. Certain enzymes display optimum activity at a particular potential, and this can be both mechanistically informative and physiologically relevant. This paper outlines the principles of protein film voltammetry by discussing some recent results from this laboratory.
蛋白质膜伏安法是研究氧化还原酶的一种相对较新的方法，其概念是将氧化还原蛋白质样品构造成电极上的薄膜，并通过各种电化学技术进行探测。酶分子以这样一种方式结合在电极表面，即存在快速电子转移，并且活性位点的化学性质在更传统的实验中能够完全保留。电极电位或催化周转的调制导致电子在酶中进出以及在酶内部移动；这被检测为一种电流，其随时间和电位以特征方式变化。从此，电位维度被引入酶动力学。存在额外的内在氧化还原中心以在埋藏的活性位点和蛋白质表面之间提供快速分子内电子转移是一个重要因素。进行协同双电子转移的中心，最明显的是黄素，会产生特别尖锐的信号，即使在光谱方法无用的情况下，作为瞬态也能被观察到。高催化活性会使电流大幅放大，即使电极上的覆盖度较低也能获得有用信息。某些酶在特定电位下显示出最佳活性，这在机理上可能具有指导意义，在生理上也可能相关。本文通过讨论本实验室的一些最新结果概述了蛋白质膜伏安法的原理。
