Redox Partner Interaction Sites in Cytochrome P450 Monooxygenases: In Silico Analysis and Experimental Validation

The native redox partners of many novel cytochrome P450 monooxygenases (CYPs) are unknown. Therefore, they are combined with non‐native redox partners to obtain catalytically active systems. Understanding the CYP‐redox partner interactions is the basis of successful protein engineering. Six redox partner interaction sites (RPISs) were identified by systematic literature, sequence, and structure analyses. All six RPISs are proposed to contribute to class II CYP‐redox partner interaction interface, whereas four and five contribute to the interaction interface in class I prokaryotic and mitochondrial CYPs, respectively. The significance of the identified RPISs was tested by designing seven variants of CYP153 A (class I) as a fusion protein with its non‐native redox partner CYP102 A1 reductase (class II) and measuring electron coupling efficiencies with a precision of 1–3 %. The best variant K166Q had an improved electron coupling efficiency of 64 % as compared to 53 % for the wild type.