We investigated electrochemical P450 vitamin D-3 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 D-3 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. (C) 2020 Elsevier Inc. All rights reserved.