单蛋白质氧化还原酶连接中催化事件的实时、无标记电转换
Real-Time, Label-free Electrical Transduction of Catalytic Events in a Single-Protein Redox Enzymatic Junction
AI总结：
本文利用电化学控制纳米隧穿连接实时无标记监测P450cam和谷胱甘肽还原酶的单蛋白催化电流切换，并验证其频率与体相转换数一致。
摘要：
单酶催化为揭示单个酶在反应过程中的动态行为提供了一种有前景的方法，可揭示在平均化集合测量中丢失的复杂异质性。在此，我们展示了对两种氧化还原酶——细胞色素P450cam和谷胱甘肽还原酶——的单蛋白质酶活性电转换进行实时、无标记监测；这些酶被捕获在浸入水性酶混合物中的电化学控制纳米级隧穿连接内。在流经单蛋白质连接的电流的各个瞬态中观察到的电导切换信号表明，隧穿电导受到酶反应的调节；化学催化过程中发生的酶氧化还原状态的细微变化导致酶连接电导率波动，并以切换信号的形式被捕获。在用于电催化的施加电化学还原电位下，每个催化循环中被捕获酶的瞬态氧化会在单蛋白质连接中打开一个额外的氧化还原介导电子隧穿通道，从而产生瞬时电流跳变，促成所观察到的电导切换特征。后者通过单蛋白质连接的电化学控制电导测量进行实验评估。对长时间捕获的切换事件进行统计分析，得到的平均频率与两种酶在标准体相测定中获得的已报道催化转换值良好相关。单酶实验揭示了酶催化显著的异质行为，并实现了单酶转换频率的定量。
英文摘要：
Single-enzyme catalysis offers a promising approach for unravelling the dynamic behaviour of individual enzymes as they undergo a reaction, revealing the complex heterogeneity that is lost in the averaged ensembles. Here we demonstrate real-time, label-free monitoring of the electrical transduction of single-protein enzymatic activity for two redox enzymes, cytochrome P450cam and glutathione reductase, trapped in an electrochemically controlled nanoscale tunnelling junction immersed in the aqueous enzymatic mixture. The conductance switching signal observed in individual transients of the electrical current flowing through the single-protein junction shows that the tunnelling conductance is modulated by the enzymatic reaction; subtle changes of the enzyme redox state occurring during the chemical catalysis process result in fluctuations of the enzyme junction conductivity, which are captured as a switching signal. At the applied electrochemical reducing potential for electrocatalysis, the transient oxidation of the trapped enzyme in every catalytic cycle opens an additional redox-mediated electron tunnelling channel in the single protein junction that results in a temporary current jump, contributing to the observed conductance switching features. The latter is experimentally assessed via electrochemically controlled conductance measurements of the single-protein junction. The statistical analysis of the switching events captured over long time periods results in average frequencies that correlate well with the reported catalytic turnover values of both enzymes obtained in standard bulk assays. The single-enzyme experiments reveal the acute heterogenous behaviour of enzymatic catalysis and the quantification of single enzyme turnover frequencies.
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