Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450_BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450_BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450_BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197–209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

P450酶的电化学体外还原是体内应用的一种有前途的替代方法。之前，我们介绍了三种工程化的P450 _BM3变体，用于苯胺羟基化，配备了碳纳米管结合肽（CNT-tag），用于在CNT电极上自组装。与野生型P450 _BM3相比，NADPH依赖性活性增强，但偶联效率仍然很低。对于P450 _BM3Verma，Schwaneberg和Roccatano（2014，Biopolymers 101，197–209）通过MD模拟计算了推定的电子转移途径（eTPs）。我们假设这些传递途径的敲除会改变系统的耦合效率。结果表明，没有针对电动P450的改进系统。但是，对于NADPH驱动的P450，使用NADPH作为还原当量，活性最高的eTP突变体显示出13倍的活性增加和32倍的偶联效率提高。这暗示了分别通过NADPH和电极还原的电子传输的替代原理。此外，该工作提出了一种改善P450与非天然底物的偶联和活性的工具。