Developing Hybrid Systems to Address O 2 Uncoupling in Multi-Component Rieske Oxygenases

Rieske non-heme iron oxygenases (ROs) are redox enzymes that are essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful in applied enzymology due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O 2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H 2 O 2 ) formation in the presence of a NADH cofactor regeneration system on the efficiency of CDO catalytic efficacy in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H 2 O 2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h at a 3-fold increase in production rate compared to CDO-WT.