Fe(III)-Superoxo σ-Addition and PCET-Driven L-DOPA 4,5-Cleavage by the Nonheme Enzyme BvDOD in the Biosynthesis Pathway of Betalamic Acid.

4,5-DOPA extradiol dioxygenase (BvDOD) catalyzes the ring cleavage of L-DOPA to generate betalamic acid. Although BvDOD shows a similar fold as those of other extradiol dioxygenases, it utilizes an atypical Fe-coordinate active site. Here, we constructed a reactant model and performed MD and QM/MM calculations to explore the reaction mechanism. Our results reveal that L-DOPA adopts a monodentate binding mode, and the two low-spin states (OS and triplet) correspond to the activated O2. During the reaction, the Fe(III)-superoxo first triggers σ-addition to C4 of L-DOPA, and then a proton-coupled electron transfer (PCET) promotes O-O cleavage. The uncoordinated phenolic hydroxyl group of the substrate plays a key role in promoting O-O scission. After the PCET event, the spin state of the system crosses to the quintet, and the lactone formation and ring opening were calculated to be quite easy. The calculated barrier at OS state (24.7 kcal/mol) is close to the estimated free energy barrier from experiment (∼23.8 kcal/mol). During the catalysis, the iron ion plays a crucial role by dramatically changing its redox state. Two key factors are necessary for the occurrence of extradiol ring cleavage: the substrate should have two adjacent phenolic hydroxyls, and the dioxygenases bind the iron cofactor with high redox activity.