We investigated electrochemical P450 vitamin D₃ hydroxylase (Vdh) reactions using promiscuous spinach ferredoxin and a small organic molecule as the mediators that shuttle electrons from the electrode to the P450. The choice of small mediator and modified electrode surface were crucially important because they decrease electron loss to undesired reactions and reduce the amount of enzyme-deactivating reactive oxygen species produced. Twice the amount of 25-hydroxyvitamin D₃ was produced under optimum conditions compared to that obtained by the conventional in vitro conversion system using NADH as the electron source, which demonstrates the electrochemical boost provided to the P450 reaction. We also confirmed that the present system works with crude extracts from Escherichia coli containing Vdh, which shows that it is possibly useful for a wide variety of P450 applications.

我们研究了使用混杂菠菜铁氧还蛋白和小的有机分子作为将电子从电极传递到P450的介体的电化学P450维生素D ₃羟化酶（Vdh）反应。选择较小的介体和修饰的电极表面至关重要，因为它们可减少因不良反应而引起的电子损失，并减少产生的使酶失活的活性氧种类。与通过使用NADH作为电子源的常规体外转化系统获得的25-羟基维生素D ₃的量相比，在最佳条件下生成了25-羟基维生素D ₃的量增加了两倍，这证明了P450反应的电化学作用。我们还确认了本系统可用于从含有Vdh的大肠杆菌，表明它可能对多种P450应用有用。