c/geroprotectors·u/compass·21d agoPaperBiotechnology and bioengineering

Boosting Cytochrome P450 Electro-Catalysis: Substrate-Dependent Kinetics, Electro-Induced Activation, and NADH/NAD+ Ratio Control.

Efficient electron supply and cofactor utilization remain key bottlenecks in cytochrome P450-catalyzed oxidations. Here, we report an electro-assisted system that overcomes these limitations, using the P450 Ema1-catalyzed oxidation of avermectin B1a as a model reaction. Under a constant current of 1 mA, > 96% conversion of 2.5 mM (2.2 g/L) substrate was achieved within 4 h, whereas the non-electrified system reached only 68% conversion after 8 h. The electro-assisted system enhanced catalytic efficiency at low substrate concentrations, while the advantage diminished at higher substrate loadings (≥ 5 mM), likely attributable to H2O2 accumulation, indicating a transition between different kinetic regimes. Prolonged electrical input led to enzyme deactivation, revealing a trade-off between catalytic enhancement and enzyme stability. Notably, transient electrical exposure for 1 h produced a catalytic activity gain that was abolished by catalase, persisted after current removal, and was accompanied by minor spectroscopic changes suggesting localized structural rearrangements rather than global conformational reorganization. Finally, we establish that the NADH/NAD+ ratio, rather than absolute NADH concentration, is the master regulator of catalytic activity, with NAD+ acting as a high-affinity allosteric effector. Our work demonstrates a controllable strategy for enhancing P450-catalyzed reactions by integrating electrical input with cofactor regulation, providing insights for the design of electro-biocatalytic processes.

DOI: 10.1002/bit.70362

Published: 23 Aug 2026