Characterisation of thermostable CYP116B reductase domains as modular electron transfer units for redox biocatalysis

Electron transfer is a common bottleneck in redox biocatalysis. Here, we report the isolation of five cytochrome P450 reductase (CPR) domains from the thermophilic, self-sufficient CYP116B family. These enzymes have considerable industrial potential because their CPR and haem domains are naturally fused, yet the catalytic properties of the individual components remain poorly understood. EPR and UV–Vis spectroscopy confirmed the canonical FMN and [2Fe–2S] cofactors were present, although occupancy varied (FMN, 30–62%; [2Fe–2S], 10–33%). The isolated CPRs were generally stable, robust and electron-transfer competent, supporting electrochemical NADPH/NADP+ oxidation/reduction and NADPH-driven reduction of ferric cytochrome c. All homologues were more thermostable than the CPR from non-thermophilic CYP116B2, with stability showing an apparent correlation with [2Fe-2S] occupancy. Separating the CPR and haem domains generally reduced the ability to perform hydroxylation on a model substrate (diclofenac). However, isolated AxCPR and AxHAEM formed a productive non-covalent combination with activity comparable to the full-length enzyme (TON 439.38 ± 5.19 versus 416.43 ± 12.11). Given the widespread use of CYP116B CPR domains as surrogate redox partners in P450 catalysis, these findings provide valuable insight into their catalytic performance. Furthermore, the results demonstrate the potential of the isolated CYP116B CPRs as modular electron-transfer units for redox biocatalysis more widely.