Cytochrome P450 enzymes (P450s) catalyze the activation of C–H bonds, followed by diverse reactions depending on the surrounding environment. To generate the active species, P450s require electrons supplied via redox partner proteins. During this process, the interaction with electron transfer proteins (ETs) not only affects electron transfer efficiency, influencing catalytic activity, but also modulates the substrate channel1, affecting reaction specificity2. Its specific but weak properties make it difficult to characterize the interaction between P450 and ETs by conventional approaches. Here, we present a proximity-based method to evaluate the P450–ET interaction.
To induce proximity, P450 and its redox partner proteins were co-anchored on a self-assembling protein scaffold. A naturally occurring heterotrimeric PCNA, whose subunits can be expressed as monomeric proteins separately but assemble into a defined trimeric complex upon mixing, serves as a powerful platform for the purpose. Even though ET binding to P450 competes with that to its reductase, this competition is negligible in a bulk system because only a small fraction of ETs are bound to either partner and most proteins are free in solution. Co-anchoring of P450 and its redox partner proteins on the PCNA scaffold brings them into spatial proximity, thereby allowing binding of the ET to P450 to effectively prevent its interaction with the reductase, resulting in a measurable decrease in ET reduction.
A significant decrease in the reduction rate of the ET by its reductase was observed upon co-anchoring. Mutations in the region of the ET indeed altered the degree of the decrease, indicating that the interaction can be distinguished using this approach. Furthermore, combining this method with immobilization onto multi-well plates simplified recombinant protein preparation, enabling high-throughput screening of the P450–ET interaction. As described above, proximity-based reduction assays provide a useful strategy for characterizing interactions between P450 and its ETs.